Comparability of comparative toxicity: insect sensitivity to imidacloprid reveals huge variations across species but also within species.

Pesticides have been identified as major drivers of insect biodiversity loss. Thus, the study of their effects on non-pest insect species has attracted a lot of attention in recent decades. In general toxicology, the 'gold standard' to assess the toxicity of a substance is to measure mass-specific LD50 (i.e. median lethal dose per unit body mass). In entomology, reviews attempting to compare these data across all available studies are lacking. To fill this gap in knowledge, we performed a systematic review of the lethality of imidacloprid for adult insects. Imidacloprid is possibly the most extensively studied insecticide in recent times, yet we found that little is comparable across studies, owing to both methodological divergence and missing estimates of body mass. By accounting for body mass whenever possible, we show how imidacloprid sensitivity spans across an apparent range of approximately six orders of magnitude across insect species. Very high variability within species can also be observed owing to differences in exposure methods and observation time. We suggest that a more comparable and comprehensive approach has both biological and economic relevance. Ultimately, this would help to identify differences that could direct research towards preventing non-target species from being negatively affected.

Acute and chronic toxicity of imidacloprid in the pollinator fly, Eristalis tenax L., assessed using a novel oral bioassay.

Imidacloprid is a neonicotinoid neurotoxin that remains widely used worldwide and persists in the environment, resulting in chronic exposure to non-target insects. To accurately map dose-dependent effects of such exposure across taxa, toxicological assays need to assess relevant modes of exposure across indicator species. However, due to the difficulty of these experiments, contact bioassays are most frequently used to quantify dose. Here, we developed a novel naturalistic feeding bioassay to precisely measure imidacloprid ingestion and its toxicity for acute and chronic exposure in a dipteran, Eristalis tenax L., an important member of an under-represented pollinator group. Flies which ingested imidacloprid dosages lower than 12.1 ng/mg all showed consistent intake volumes and learned improved feeding efficiency over successive feeding sessions. In contrast, at doses of 12.1 ng/mg and higher flies showed a rapid onset of severe locomotive impairment which prevented them from completing the feeding task. Neither probability of survival nor severe locomotive impairment were significantly higher than the control group until doses of 1.43 ng/mg or higher were reached. We were unable to measure a median lethal dose for acute exposure (72 h) due to flies possessing a relatively high tolerance for imidacloprid. However, with chronic exposure (18 days), mortality went up and an LD50 of 0.41 ng/mg was estimated. Severe locomotive impairment (immobilisation) tended to occur earlier and at lower dosages than lethality, with ED50s of 7.82 ng/mg and 0.17 ng/mg for acute and chronic exposure, respectively. We conclude that adult Eristalis possess a much higher tolerance to this toxin than the honeybees that they mimic. The similarity of the LD50 to other dipterans such as the fruitfly and the housefly suggests that there may be a phylogenetic component to pesticide tolerance that merits further investigation. The absence of obvious adverse effects at sublethal dosages also underscores a need to develop better tools for quantifying animal behaviour to evaluate the impact of insecticides on foraging efficiency in economically important species.

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.

A Target-Detecting Visual Neuron in the Dragonfly Locks on to Selectively Attended Targets.

The visual world projects a complex and rapidly changing image onto the retina of many animal species. This presents computational challenges for those animals reliant on visual processing to provide an accurate representation of the world. One such challenge is parsing a visual scene for the most salient targets, such as the selection of prey amid a swarm. The ability to selectively prioritize processing of some stimuli over others is known as 'selective attention'. We recently identified a dragonfly visual neuron called 'Centrifugal Small Target Motion Detector 1' (CSTMD1) that exhibits selective attention when presented with multiple, equally salient targets. Here we conducted in vivo, electrophysiological recordings from CSTMD1 in wild-caught male dragonflies (Hemicordulia tau), while presenting visual stimuli on an LCD monitor. To identify the target selected in any given trial, we uniquely modulated the intensity of the moving targets (frequency tagging). We found that the frequency information of the selected target is preserved in the neuronal response, while the distracter is completely ignored. We also show that the competitive system that underlies selection in this neuron can be biased by the presentation of a preceding target on the same trajectory, even when it is of lower contrast than an abrupt, novel distracter. With this improved method for identifying and biasing target selection in CSTMD1, the dragonfly provides an ideal animal model system to probe the neuronal mechanisms underlying selective attention.SIGNIFICANCE STATEMENT We present the first application of frequency tagging to intracellular neuronal recordings, demonstrating that the frequency component of a stimulus is encoded in the spiking response of an individual neuron. Using this technique as an identifier, we demonstrate that CSTMD1 'locks on' to a selected target and encodes the absolute strength of this target, even in the presence of abruptly appearing, high-contrast distracters. The underlying mechanism also permits the selection mechanism to switch between targets mid-trial, even among equivalent targets. Together, these results demonstrate greater complexity in this selective attention system than would be expected in a winner-takes-all network. These results are in contrast to typical findings in the primate and avian brain, but display intriguing resemblance to observations in human psychophysics.

Properties of predictive gain modulation in a dragonfly visual neuron.

Dragonflies pursue and capture tiny prey and conspecifics with extremely high success rates. These moving targets represent a small visual signal on the retina and successful chases require accurate detection and amplification by downstream neuronal circuits. This amplification has been observed in a population of neurons called small target motion detectors (STMDs), through a mechanism we term predictive gain modulation. As targets drift through the neuron's receptive field, spike frequency builds slowly over time. This increased likelihood of spiking or gain is modulated across the receptive field, enhancing sensitivity just ahead of the target's path, with suppression of activity in the remaining surround. Whilst some properties of this mechanism have been described, it is not yet known which stimulus parameters modulate the amount of response gain. Previous work suggested that the strength of gain enhancement was predominantly determined by the duration of the target's prior path. Here, we show that predictive gain modulation is more than a slow build-up of responses over time. Rather, the strength of gain is dependent on the velocity of a prior stimulus combined with the current stimulus attributes (e.g. angular size). We also describe response variability as a major challenge of target-detecting neurons and propose that the role of predictive gain modulation is to drive neurons towards response saturation, thus minimising neuronal variability despite noisy visual input signals.

Photoreceptor signalling is sufficient to explain the detectability threshold of insect aerial pursuers.

An essential biological task for many flying insects is the detection of small, moving targets, such as when pursuing prey or conspecifics. Neural pathways underlying such 'target-detecting' behaviours have been investigated for their sensitivity and tuning properties (size, velocity). However, which stage of neuronal processing limits target detection is not yet known. Here, we investigated several skilled, aerial pursuers (males of four insect species), measuring the target-detection limit (signal-to-noise ratio) of light-adapted photoreceptors. We recorded intracellular responses to moving targets of varying size, extended well below the nominal resolution of single ommatidia. We found that the signal detection limit (2× photoreceptor noise) matches physiological or behavioural target-detection thresholds observed in each species. Thus, across a diverse range of flying insects, individual photoreceptor responses to changes in light intensity establish the sensitivity of the feature detection pathway, indicating later stages of processing are dedicated to feature tuning, tracking and selection.

Three-dimensional functional human neuronal networks in uncompressed low-density electrospun fiber scaffolds.

We demonstrate an artificial three-dimensional (3D) electrical active human neuronal network system, by the growth of brain neural progenitors in highly porous low density electrospun poly-ε-caprolactone (PCL) fiber scaffolds. In neuroscience research cell-based assays are important experimental instruments for studying neuronal function in health and disease. Traditional cell culture at 2D-surfaces induces abnormal cell-cell contacts and network formation. Hence, there is a tremendous need to explore in vivo-resembling 3D neural cell culture approaches. We present an improved electrospinning method for fabrication of scaffolds that promote neuronal differentiation into highly 3D integrated networks, formation of inhibitory and excitatory synapses and extensive neurite growth. Notably, in 3D scaffolds in vivo-resembling intermixed neuronal and glial cell network were formed, whereas in parallel 2D cultures a neuronal cell layer grew separated from an underlying glial cell layer. Hence, the use of the 3D cell assay presented will most likely provide more physiological relevant results.

Correlation between OFF and ON channels underlies dark target selectivity in an insect visual system.

In both vertebrates and invertebrates, evidence supports separation of luminance increments and decrements (ON and OFF channels) in early stages of visual processing (Hartline, 1938; Joesch et al., 2010); however, less is known about how these parallel pathways are recombined to encode form and motion. In Drosophila, genetic knockdown of inputs to putative ON and OFF pathways and direct recording from downstream neurons in the wide-field motion pathway reveal that local elementary motion detectors exist in pairs that separately correlate contrast polarity channels, ON with ON and OFF with OFF (Joesch et al., 2013). However, behavioral responses to reverse-phi motion of discrete features reveal additional correlations of the opposite signs (Clark et al., 2011). We here present intracellular recordings from feature detecting neurons in the dragonfly that provide direct physiological evidence for the correlation of OFF and ON pathways. These neurons show clear polarity selectivity for feature contrast, responding strongly to targets that are darker than the background and only weakly to dark contrasting edges. These dark target responses are much stronger than the linear combination of responses to ON and OFF edges. We compare these data with output from elementary motion detector-based models (Eichner et al., 2011; Clark et al., 2011), with and without stages of strong center-surround antagonism. Our data support an alternative elementary small target motion detector model, which derives dark target selectivity from the correlation of a delayed OFF with an un-delayed ON signal at each individual visual processing unit (Wiederman et al., 2008, 2009).

Discrimination of features in natural scenes by a dragonfly neuron.

Flying insects engage in spectacular high-speed pursuit of targets, requiring visual discrimination of moving objects against cluttered backgrounds. As a first step toward understanding the neural basis for this complex task, we used computational modeling of insect small target motion detector (STMD) neurons to predict responses to features within natural scenes and then compared this with responses recorded from an identified STMD neuron in the dragonfly brain (Hemicordulia tau). A surprising model prediction confirmed by our electrophysiological recordings is that even heavily cluttered scenes contain very few features that excite these neurons, due largely to their exquisite tuning for small features. We also show that very subtle manipulations of the image cause dramatic changes in the response of this neuron, because of the complex inhibitory and facilitatory interactions within the receptive field.

Spontaneous Cell Cluster Formation in Human iPSC-Derived Neuronal Spheroid Networks Influences Network Activity.

Three-dimensional neuronal culture systems such as spheroids, organoids, and assembloids constitute a branch of neuronal tissue engineering that has improved our ability to model the human brain in the laboratory. However, the more elaborate the brain model, the more difficult it becomes to study functional properties such as electrical activity at the neuronal level, similar to the challenges of studying neurophysiology in vivo We describe a simple approach to generate self-assembled three-dimensional neuronal spheroid networks with defined human cell composition on microelectrode arrays. Such spheroid networks develop a highly three-dimensional morphology with cell clusters up to 60 µm in thickness and are interconnected by pronounced bundles of neuronal fibers and glial processes. We could reliably record from up to hundreds of neurons simultaneously per culture for ≤90 d. By quantifying the formation of these three-dimensional structures over time, while regularly monitoring electrical activity, we were able to establish a strong link between spheroid morphology and network activity. In particular, the formation of cell clusters accelerates formation and maturation of correlated network activity. Astrocytes both influence electrophysiological network activity as well as accelerate the transition from single cell layers to cluster formation. Higher concentrations of astrocytes also have a strong effect of modulating synchronized network activity. This approach thus represents a practical alternative to often complex and heterogeneous organoids, providing easy access to activity within a brain-like 3D environment.Significance StatementNeuronal "organoid" cultures with multiple cell types grown on elaborate three-dimensional scaffolds have become popular tools to generate brain-like properties in vitro but bring with them similar problems concerning access to physiological function as real brain tissue. Here, we developed a new approach to form simple brain-like spheroid networks from human neurons, but using the normal supporting cells of the brain, astrocytes, as the scaffold. By growing these cultures on conventional microelectrode arrays, we were able to observe development of complex patterns of electrical activity for months. Our results highlight how formation of three-dimensional structures accelerated the formation of synchronized neuronal network activity and provide a promising new simple model system for studying interactions between known human cell types in vitro.

Dragonfly Neurons Selectively Attend to Targets Within Natural Scenes.

Aerial predators, such as the dragonfly, determine the position and movement of their prey even when both are moving through complex, natural scenes. This task is likely supported by a group of neurons in the optic lobe which respond to moving targets that subtend less than a few degrees. These Small Target Motion Detector (STMD) neurons are tuned to both target size and velocity, whilst also exhibiting facilitated responses to targets traveling along continuous trajectories. When presented with a pair of targets, some STMDs generate spiking activity that represent a competitive selection of one target, as if the alternative does not exist (i.e., selective attention). Here, we describe intracellular responses of CSTMD1 (an identified STMD) to the visual presentation of targets embedded within cluttered, natural scenes. We examine CSTMD1 response changes to target contrast, as well as a range of target and background velocities. We find that background motion affects CSTMD1 responses via the competitive selection between features within the natural scene. Here, robust discrimination of our artificially embedded "target" is limited to scenarios when its velocity is matched to, or greater than, the background velocity. Additionally, the background's direction of motion affects discriminability, though not in the manner observed in STMDs of other flying insects. Our results highlight that CSTMD1's competitive responses are to those features best matched to the neuron's underlying spatiotemporal tuning, whether from the embedded target or other features in the background clutter. In many scenarios, CSTMD1 responds robustly to targets moving through cluttered scenes. However, whether this neuronal system could underlie the task of competitively selecting slow moving prey against fast-moving backgrounds remains an open question.

Preattentive facilitation of target trajectories in a dragonfly visual neuron.

The ability to pursue targets in visually cluttered and distraction-rich environments is critical for predators such as dragonflies. Previously, we identified Centrifugal Small-Target Motion Detector 1 (CSTMD1), a dragonfly visual neuron likely involved in such target-tracking behaviour. CSTMD1 exhibits facilitated responses to targets moving along a continuous trajectory. Moreover, CSTMD1 competitively selects a single target out of a pair. Here, we conducted in vivo, intracellular recordings from CSTMD1 to examine the interplay between facilitation and selection, in response to the presentation of paired targets. We find that neuronal responses to both individual trajectories of simultaneous, paired targets are facilitated, rather than being constrained to the single, selected target. Additionally, switches in selection elicit suppression which is likely an important attribute underlying target pursuit. However, binocular experiments reveal these results are constrained to paired targets within the same visual hemifield, while selection of a target in one visual hemifield establishes ocular dominance that prevents facilitation or response to contralaterally presented targets. These results reveal that the dragonfly brain preattentively represents more than one target trajectory, to balance between attentional flexibility and resistance against distraction.

Sexual dimorphism in the hoverfly motion vision pathway.

Many insects perform high-speed aerial maneuvers in which they navigate through visually complex surrounds. Among insects, hoverflies stand out, with males switching from stationary hovering to high-speed pursuit at extreme angular velocities [1]. In dipterans, 50-60 large interneurons -- the lobula-plate tangential cells (LPTCs) -- detect changes in optic flow experienced during flight [2-5]. It has been predicted that large LPTC receptive fields are a requirement of accurate "matched filters" of optic flow [6]. Whereas many fly taxa have three horizontal system (HS) LPTC neurons in each hemisphere, hoverflies have four [7], possibly reflecting the more sophisticated flight behavior. We here show that the most dorsal hoverfly neuron (HS north [HSN]) is sexually dimorphic, with the male receptive field substantially smaller than in females or in either sex of blowflies. The (hoverfly-specific) HSN equatorial (HSNE) is, however, sexually isomorphic. Using complex optic flow, we show that HSN, despite its smaller receptive field, codes yaw velocity as well as HSNE. Responses to a target moving against a plain or textured background suggest that the male HSN could potentially play a role in target pursuit under some conditions.

Rapid contrast gain reduction following motion adaptation.

Neural and sensory systems adapt to prolonged stimulation to allow signaling across broader input ranges than otherwise possible with the limited bandwidth of single neurons and receptors. In the visual system, adaptation takes place at every stage of processing, from the photoreceptors that adapt to prevailing luminance conditions, to higher-order motion-sensitive neurons that adapt to prolonged exposure to motion. Recent experiments using dynamic, fluctuating visual stimuli indicate that adaptation operates on a time scale similar to that of the response itself. Further work from our own laboratory has highlighted the role for rapid motion adaptation in reliable encoding of natural image motion. Physiologically, motion adaptation can be broken down into four separate components. It is not clear from the previous studies which of these motion adaptation components are involved in the fast and dynamic response changes. To investigate the adapted response in more detail, we therefore analyzed the effect of motion adaptation using a test-adapt-test protocol with adapting durations ranging from 20 ms to 20 s. Our results underscore the very rapid rate of motion adaptation, suggesting that under free flight, visual motion-sensitive neurons continuously adapt to the changing scenery. This might help explain recent observations of strong invariance in the response to natural scenes with highly variable contrast and image structure.

Spatial facilitation by a high-performance dragonfly target-detecting neuron.

Many animals visualize and track small moving targets at long distances-be they prey, approaching predators or conspecifics. Insects are an excellent model system for investigating the neural mechanisms that have evolved for this challenging task. Specialized small target motion detector (STMD) neurons in the optic lobes of the insect brain respond strongly even when the target size is below the resolution limit of the eye. Many STMDs also respond robustly to small targets against complex stationary or moving backgrounds. We hypothesized that this requires a complex mechanism to avoid breakthrough responses by background features, and yet to adequately amplify the weak signal of tiny targets. We compared responses of dragonfly STMD neurons to small targets that begin moving within the receptive field with responses to targets that approach the same location along longer trajectories. We find that responses along longer trajectories are strongly facilitated by a mechanism that builds up slowly over several hundred milliseconds. This allows the neurons to give sustained responses to continuous target motion, thus providing a possible explanation for their extraordinary sensitivity.

Local and global responses of insect motion detectors to the spatial structure of natural scenes.

As a consequence of the non-linear correlation mechanism underlying motion detection, the variability in local pattern structure and contrast inherent within natural scenes profoundly influences local motion responses. To accurately interpret optic flow induced by self-motion, neurons in many dipteran flies smooth this "pattern noise" by wide-field spatial integration. We investigated the role that size and shape of the receptive field plays in smoothing out pattern noise in two unusual hoverfly optic flow neurons: one (HSN) with an exceptionally small receptive field and one (HSNE) with a larger receptive field. We compared the local and global responses to a sequence of panoramic natural images in these two neurons with a parsimonious model for elementary motion detection weighted for their spatial receptive fields. Combined with manipulation of size and contrast of the stimulus images, this allowed us to separate spatial integration properties arising from the receptive field, from other local and global non-linearities, such as motion adaptation and dendritic gain control. We show that receptive field properties alone are poor predictors of the response to natural scenes. If anything, additional non-linearity enhances the pattern dependence of HSN's response, particularly to vertically elongated features, suggesting that it may serve a role in forward fixation during hovering.

Acute Application of Imidacloprid Alters the Sensitivity of Direction Selective Motion Detecting Neurons in an Insect Pollinator.

Cholinergic pesticides, such as the neonicotinoid imidacloprid, are the most important insecticides used for plant protection worldwide. In recent decades, concerns have been raised about side effects on non-target insect species, including altered foraging behavior and navigation. Although pollinators rely on visual cues to forage and navigate their environment, the effects of neonicotinoids on visual processing have been largely overlooked. To test the effect of acute treatment with imidacloprid at known concentrations in the brain, we developed a modified electrophysiological setup that allows recordings of visually evoked responses while perfusing the brain in vivo. We obtained long-lasting recordings from direction selective wide-field, motion sensitive neurons of the hoverfly pollinator, Eristalis tenax. Neurons were treated with imidacloprid (3.9 µM, 0.39 µM or a sham control treatment using the solvent (dimethylsulfoxide) only. Exposure to a high, yet sub-lethal concentration of imidacloprid significantly alters their physiological response to motion stimuli. We observed a general effect of imidacloprid (3.9 µM) increasing spontaneous activity, reducing contrast sensitivity and giving weaker directional tuning to wide-field moving stimuli, with likely implications for errors in flight control, hovering and routing. Our electrophysiological approach reveals the robustness of the fly visual pathway against cholinergic perturbance (i.e., at 0.39 µM) but also potential threatening effects of cholinergic pesticides (i.e., evident at 3.9 µM) for the visual motion detecting system of an important pollinator.

A new, fluorescence-based method for visualizing the pseudopupil and assessing optical acuity in the dark compound eyes of honeybees and other insects.

Recent interest in applying novel imaging techniques to infer optical resolution in compound eyes underscores the difficulty of obtaining direct measures of acuity. A widely used technique exploits the principal pseudopupil, a dark spot on the eye surface representing the ommatidial gaze direction and the number of detector units (ommatidia) viewing that gaze direction. However, dark-pigmented eyes, like those of honeybees, lack a visible pseudopupil. Attempts over almost a century to estimate optical acuity in this species are still debated. Here, we developed a method to visualize a stable, reliable pseudopupil by staining the photoreceptors with fluorescent dyes. We validated this method in several species and found it to outperform the dark pseudopupil for this purpose, even in pale eyes, allowing more precise location of the gaze centre. We then applied this method to estimate the sampling resolution in the frontal part of the eye of the honeybee forager. We found a broad frontal acute zone with interommatidial angles below 2° and a minimum interommatidial angle of 1.3°, a broader, sharper frontal acute zone than previously reported. Our study provides a new method to directly measure the sampling resolution in most compound eyes of living animals.

Modeling Nonlinear Dendritic Processing of Facilitation in a Dragonfly Target-Tracking Neuron.

Dragonflies are highly skilled and successful aerial predators that are even capable of selectively attending to one target within a swarm. Detection and tracking of prey is likely to be driven by small target motion detector (STMD) neurons identified from several insect groups. Prior work has shown that dragonfly STMD responses are facilitated by targets moving on a continuous path, enhancing the response gain at the present and predicted future location of targets. In this study, we combined detailed morphological data with computational modeling to test whether a combination of dendritic morphology and nonlinear properties of NMDA receptors could explain these observations. We developed a hybrid computational model of neurons within the dragonfly optic lobe, which integrates numerical and morphological components. The model was able to generate potent facilitation for targets moving on continuous trajectories, including a localized spotlight of maximal sensitivity close to the last seen target location, as also measured during in vivo recordings. The model did not, however, include a mechanism capable of producing a traveling or spreading wave of facilitation. Our data support a strong role for the high dendritic density seen in the dragonfly neuron in enhancing non-linear facilitation. An alternative model based on the morphology of an unrelated type of motion processing neuron from a dipteran fly required more than three times higher synaptic gain in order to elicit similar levels of facilitation, despite having only 20% fewer synapses. Our data support a potential role for NMDA receptors in target tracking and also demonstrate the feasibility of combining biologically plausible dendritic computations with more abstract computational models for basic processing as used in earlier studies.

Local and large-range inhibition in feature detection.

Lateral inhibition is perhaps the most ubiquitous of neuronal mechanisms, having been demonstrated in early stages of processing in many different sensory pathways of both mammals and invertebrates. Recent work challenges the long-standing view that assumes that similar mechanisms operate to tune neuronal responses to higher order properties. Scant evidence for lateral inhibition exists beyond the level of the most peripheral stages of visual processing, leading to suggestions that many features of the tuning of higher order visual neurons can be accounted for by the receptive field and other intrinsic coding properties of visual neurons. Using insect target neurons as a model, we present unequivocal evidence that feature tuning is shaped not by intrinsic properties but by potent spatial lateral inhibition operating well beyond the first stages of visual processing. In addition, we present evidence for a second form of higher-order spatial inhibition--a long-range interocular transfer of information that we argue serves a role in establishing interocular rivalry and thus potentially a neural substrate for directing attention to single targets in the presence of distracters. In so doing, we demonstrate not just one, but two levels of spatial inhibition acting beyond the level of peripheral processing.