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An important step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. Here, we use in vivo two-photon imaging of genetically encoded voltage and calcium indicators, ArcLight and GCaMP6f, respectively, to measure responses in direction-selective T4 neurons of femaleDrosophila. Comparison between ArcLight and GCaMP6f signals reveals calcium signals to have a significantly higher direction selectivity compared with voltage signals. Using these recordings, we build a model which transforms T4 voltage responses into calcium responses. Using a cascade of thresholding, temporal filtering and a stationary nonlinearity, the model reproduces experimentally measured calcium responses across different visual stimuli. These findings provide a mechanistic underpinning of the voltage to calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons. Measuring the directional tuning of postsynaptic vertical system (VS)-cells with inputs from other cells blocked, we found that, indeed, it matches the one of the calcium signal in presynaptic T4 cells.SIGNIFICANCE STATEMENTThe transformation of voltage to calcium influx is an important step in the signaling cascade within a nerve cell. While this process has been intensely studied in the context of transmitter release mechanism, its consequences for information transmission and neural computation are unclear. Here, we measured both membrane voltage and cytosolic calcium levels in direction-selective cells ofDrosophilain response to a large set of visual stimuli. We found direction selectivity in the calcium signal to be significantly enhanced compared with membrane voltage through a nonlinear transformation of voltage to calcium. Our findings highlight the importance of an additional step in the signaling cascade for information processing within single nerve cells.

The ability to drive expression of exogenous genes in different tissues and cell types, under the control of specific enhancers, has been crucial for discovery in biology. While many enhancers drive expression broadly, several genetic tools were developed to obtain access to isolated cell types. Studies of spatially organized neuropiles in the central nervous system of fruit flies have raised the need for a system that targets subsets of cells within a single neuronal type, a feat currently dependent on stochastic flip-out methods. To access the same cells within a given expression pattern consistently across fruit flies, we developed the light-gated expression system LOV-LexA. We combined the bacterial LexA transcription factor with the plant-derived light, oxygen, or voltage photosensitive domain and a fluorescent protein. Exposure to blue light uncages a nuclear localizing signal in the C-terminal of the light, oxygen, or voltage domain and leads to the translocation of LOV-LexA to the nucleus, with the subsequent initiation of transcription. LOV-LexA enables spatial and temporal control of expression of transgenes under LexAop sequences in larval fat body and pupal and adult neurons with blue light. The LOV-LexA tool is ready to use with GAL4 and Split-GAL4 drivers in its current form and constitutes another layer of intersectional genetics that provides light-controlled genetic access to specific cells across flies.

A critical step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. Here, we use in vivo two-photon imaging of genetically encoded voltage and calcium indicators, Arclight and GCaMP6f respectively, to measure responses in Drosophila direction-selective T4 neurons.Comparison between Arclight and GCaMP6f signals revealed calcium signals to have a significantly higher direction selectivity compared to voltage signals. Using these recordings we build a model which transforms T4 voltage responses to calcium responses. The model reproduces experimentally measured calcium responses across different visual stimuli using different temporal filtering steps and a stationary non-linearity. These findings provide a mechanistic underpinning of the voltage-to-calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons.

Nonlinear, multiplication-like operations carried out by individual nerve cells greatly enhance the computational power of a neural system1–3, but our understanding of their biophysical implementation is scant. Here we pursue this problem in the Drosophila melanogaster ON motion vision circuit4,5, in which we record the membrane potentials of direction-selective T4 neurons and of their columnar input elements6,7 in response to visual and pharmacological stimuli in vivo. Our electrophysiological measurements and conductance-based simulations provide evidence for a passive supralinear interaction between two distinct types of synapse on T4 dendrites. We show that this multiplication-like nonlinearity arises from the coincidence of cholinergic excitation and release from glutamatergic inhibition. The latter depends on the expression of the glutamate-gated chloride channel GluClα8,9 in T4 neurons, which sharpens the directional tuning of the cells and shapes the optomotor behaviour of the animals. Interacting pairs of shunting inhibitory and excitatory synapses have long been postulated as an analogue approximation of a multiplication, which is integral to theories of motion detection10,11, sound localization12 and sensorimotor control13.

Nonlinear, multiplication-like operations carried out by individual nerve cells greatly enhance the computational power of a neural system

Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1–R6 through Tm3 and Mi1 is sufficient to confer Dm8’s UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1–R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8’s spatial opponency and animals’ phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.

Electrical synapses are present in almost all organisms that have a nervous system. However, their brain-wide expression patterns and the full range of contributions to neural function are unknown in most species. Here, we first provide a light-microscopic, immunohistochemistry-based anatomical map of all innexin gap junction proteins-the building blocks of electrical synapses-in the central nervous system of Drosophila melanogaster. Of those innexin types that are expressed in the nervous system, some localize to glial cells, whereas others are predominantly expressed in neurons, with shakB being the most widely expressed neuronal innexin. We then focus on the function of shakB in VS/HS cells-a class of visual projection neurons-thereby uncovering an unexpected role for electrical synapses. Removing shakB from these neurons leads to spontaneous, cell-autonomous voltage and calcium oscillations, demonstrating that electrical synapses are required for these cells' intrinsic stability. Furthermore, we investigate the role of shakB-type electrical synapses in early visual processing. We find that the loss of shakB from the visual circuits upstream of VS/HS cells differentially impairs ON and OFF visual motion processing pathways but is not required for the computation of direction selectivity per se. Taken together, our study demonstrates that electrical synapses are widespread across the Drosophila nervous system and that they play essential roles in neuronal function and visual information processing.

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.

Visual motion detection is among the best understood neuronal computations. One assumed behavioural role is to detect self-motion and to counteract involuntary course deviations, extensively investigated in tethered walking or flying flies. In free flight, however, any deviation from a straight course is signalled by both the visual system as well as by proprioceptive mechanoreceptors called ‘halteres’, which are the equivalent of the vestibular system in vertebrates. Therefore, it is yet unclear to what extent motion vision contributes to course control, or whether straight flight is completely controlled by proprioceptive feedback from the halteres. To answer these questions, 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 difficulties maintaining a straight flight trajectory, much like control flies in the dark. By unilateral wing clipping, we generated an asymmetry in propulsory 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 unequivocally demonstrate that motion vision is necessary to fly straight under realistic conditions.

Summary: For a proper understanding of neural circuit function, it is important to know which signals neurons relay to their downstream partners. Calcium imaging with genetically encoded calcium sensors like GCaMP has become the default approach for mapping these responses. How well such measurements represent the true neurotransmitter output of any given cell, however, remains unclear. Here, we demonstrate the viability of the glutamate sensor iGluSnFR for 2-photon in vivo imaging in Drosophila melanogaster and prove its usefulness for estimating spatiotemporal receptive fields in the visual system. We compare the results obtained with iGluSnFR with the ones obtained with GCaMP6f and find that the spatial aspects of the receptive fields are preserved between indicators. In the temporal domain, however, measurements obtained with iGluSnFR reveal the underlying response properties to be much faster than those acquired with GCaMP6f. Our approach thus offers a more accurate description of glutamatergic neurons in the fruit fly. : Optical Imaging; Sensory Neuroscience; Techniques in Neuroscience Subject Areas: Optical Imaging, Sensory Neuroscience, Techniques in Neuroscience

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., Summary: Morphological and transcriptomic analyses allowed the identification of a combinatorial code of transcription factors that controls the development of subtype-specific morphologies in motion-detecting neurons of the Drosophila visual system.

Vision is an important sensory modality for navigation in roaming animals. In contrast to most vertebrates, insects usually must cope with low resolution retinal images and the inability to infer spatial features using accommodation or stereovision. However, during locomotion, the retinal input is dominated by characteristic panoramic image shifts, termed optic flow, that depend on self-motion parameters and environmental features. Therefore, optic flow provides a rich source of information guiding locomotion speed as well as the position and orientation of animals over time relative to their surroundings. Here, focusing on flight behavior, we describe the strategies and putative underlying neuronal mechanisms by which insects control their course through processing of visual motion cues.

Approaches are sought after to regulate ionotropic and chronotropic properties of the mammalian heart. Electrodes are commonly used for rapidly exciting cardiac tissue and resetting abnormal pacing. With the advent of optogenetics and the use of tissue-specific expression of light-activated channels, cardiac cells cannot only be excited but also inhibited with ion-selective conductance. As a proof of concept for the ability to slow down cardiac pacing, anion-conducting channelrhodopsins (GtACR1/2) and the anion pump halorhodopsin (eNpHR) were expressed in hearts of larval Drosophila and activated by light. Unlike body wall muscles in most animals, the equilibrium potential for Cl− is more positive as compared to the resting membrane potential in larval Drosophila. As a consequence, upon activating the two forms of GtACR1 and 2 with low light intensity the heart rate increased, likely due to depolarization and opening of voltage-gated Ca2+ channels. However, with very intense light activation the heart rate ceases, which may be due to Cl– shunting to the reversal potential for chloride. Activating eNpHR hyperpolarizes body wall and cardiac muscle in larval Drosophila and rapidly decreases heart rate. The decrease in heart rate is related to light intensity. Intense light activation of eNpHR stops the heart from beating, whereas lower intensities slowed the rate. Even with upregulation of the heart rate with serotonin, the pacing of the heart was slowed with light. Thus, regulation of the heart rate in Drosophila can be accomplished by activating anion-conducting channelrhodopsins using light. These approaches are demonstrated in a genetically amenable insect model.

The visual system must make predictions to compensate for inherent delays in its processing. Yet little is known, mechanistically, about how prediction aids natural behaviors. Here, we show that despite a 20-30ms intrinsic processing delay, the vertical motion sensitive (VS) network of the blowfly achieves maximally efficient prediction. This prediction enables the fly to fine-tune its complex, yet brief, evasive flight maneuvers according to its initial ego-rotation at the time of detection of the visual threat. Combining a rich database of behavioral recordings with detailed compartmental modeling of the VS network, we further show that the VS network has axonal gap junctions that are critical for optimal prediction. During evasive maneuvers, a VS subpopulation that directly innervates the neck motor center can convey predictive information about the fly’s future ego-rotation, potentially crucial for ongoing flight control. These results suggest a novel sensory-motor pathway that links sensory prediction to behavior., Author summary Survival-critical behaviors shape neural circuits to translate sensory information into strikingly fast predictions, e.g. in escaping from a predator faster than the system’s processing delay. We show that the fly visual system implements fast and accurate prediction of its visual experience. This provides crucial information for directing fast evasive maneuvers that unfold over just 40ms. Our work shows how this fast prediction is implemented, mechanistically, and suggests the existence of a novel sensory-motor pathway from the fly visual system to a wing steering motor neuron. Echoing and amplifying previous work in the retina, our work hypothesizes that the efficient encoding of predictive information is a universal design principle supporting fast, natural behaviors.

The reliable estimation of motion across varied surroundings represents a survival-critical task for sighted animals. How neural circuits have adapted to the particular demands of natural environments, however, is not well understood. We explored this question in the visual system of Drosophila melanogaster. Here, as in many mammalian retinas, motion is computed in parallel streams for brightness increments (ON) and decrements (OFF). When genetically isolated, ON and OFF pathways proved equally capable of accurately matching walking responses to realistic motion. To our surprise, detailed characterization of their functional tuning properties through in vivo calcium imaging and electrophysiology revealed stark differences in temporal tuning between ON and OFF channels. We trained an in silico motion estimation model on natural scenes and discovered that our optimized detector exhibited differences similar to those of the biological system. Thus, functional ON-OFF asymmetries in fly visual circuitry may reflect ON-OFF asymmetries in natural environments.

Estimating motion is a fundamental task for the visual system of sighted animals. In Drosophila, direction-selective T4 and T5 cells respond to moving brightness increments (ON) and decrements (OFF), respectively. Current algorithmic models of the circuit are based on the interaction of two differentially filtered signals. However, electron microscopy studies have shown that T5 cells receive their major input from four classes of neurons: Tm1, Tm2, Tm4, and Tm9. Using two-photon calcium imaging, we demonstrate that T5 is the first direction-selective stage within the OFF pathway. The four cells provide an array of spatiotemporal filters to T5. Silencing their synaptic output in various combinations, we find that all input elements are involved in OFF motion detection to varying degrees. Our comprehensive survey challenges the simplified view of how neural systems compute the direction of motion and suggests that an intricate interplay of many signals results in direction selectivity.

A neuron is conventionally regarded as a single processing unit. It receives input from one or several presynaptic cells, transforms these signals, and transmits one output signal to its postsynaptic partners. Exceptions exist: amacrine cells in the mamma- lian retina [1-3] or interneurons in the locust meso- thoracic ganglion [4] are thought to represent many electrically isolated microcircuits within one neuron. An extreme case of such an amacrine cell has recently been described in the Drosophila visual system. This cell, called CT1, reaches into two neuropils of the optic lobe, where it visits each of 700 repetitive columns, thereby covering the whole visual field [5, 6]. Due to its unusual morphology, CT1 has been suspected to perform local computations [6, 7], but this has never been proven. Using 2-photon calcium imaging and visual stimulation, we find highly compartmentalized retinotopic response properties in neighboring terminals of CT1, with each terminal acting as an independent functional unit. Model simulations demonstrate that this extreme case of compartmentalization is at the biophysical limit of neural computation.

In the Drosophila visual system, T4/T5 neurons represent the first stage of computation of the direction of visual motion. T4 and T5 neurons exist in four subtypes, each responding to motion in one of the four cardinal directions and projecting axons into one of the four lobula plate layers. However, all T4/T5 neurons share properties essential for sensing motion. How T4/T5 neurons acquire their properties during development is poorly understood. We reveal that the transcription factors SoxN and Sox102F control the acquisition of properties common to all T4/T5 neuron subtypes, i.e. the layer specificity of dendrites and axons. Accordingly, adult flies are motion blind after disruption of SoxN or Sox102F in maturing T4/T5 neurons. We further find that the transcription factors Ato and Dac are redundantly required in T4/T5 neuron progenitors for SoxN and Sox102F expression in T4/T5 neurons, linking the transcriptional programmes specifying progenitor identity to those regulating the acquisition of morphological properties in neurons. Our work will help to link structure, function and development in a neuronal type performing a computation that is conserved across vertebrate and invertebrate visual systems., Summary: RNA interference identifies SoxN and Sox102F as key regulators of the morphological properties of a neuronal type of the fruit fly visual system that computes the direction of motion.

SummaryWhen navigating in their environment, animals use visual motion cues as feedback signals that are elicited by their own motion. Such signals are provided by wide-field neurons sampling motion directions at multiple image points as the animal maneuvers. Each one of these neurons responds selectively to a specific optic flow-field representing the spatial distribution of motion vectors on the retina. Here, we describe the discovery of a group of local, inhibitory interneurons in the fruit fly Drosophila key for filtering these cues. Using anatomy, molecular characterization, activity manipulation, and physiological recordings, we demonstrate that these interneurons convey direction-selective inhibition to wide-field neurons with opposite preferred direction and provide evidence for how their connectivity enables the computation required for integrating opposing motions. Our results indicate that, rather than sharpening directional selectivity per se, these circuit elements reduce noise by eliminating non-specific responses to complex visual information.

The fly visual system offers a unique opportunity to explore computations performed by single neurons. Two previous studies characterized, in vivo, the receptive field (RF) of the vertical system (VS) cells of the blowfly (calliphora vicina), both intracellularly in the axon, and, independently using Ca2+ imaging, in hundreds of distal dendritic branchlets. We integrated this information into detailed passive cable and compartmental models of 3D reconstructed VS cells. Within a given VS cell type, the transfer resistance (TR) from different branchlets to the axon differs substantially, suggesting that they contribute unequally to the shaping of the axonal RF. Weighting the local RFs of all dendritic branchlets by their respective TR yielded a faithful reproduction of the axonal RF. The model also predicted that the various dendritic branchlets are electrically decoupled from each other, thus acting as independent local functional subunits. The study suggests that single neurons in the fly visual system filter dendritic noise and compute the weighted average of their inputs.

Optogenetic channels and ion pumps have become indispensable tools in neuroscience to manipulate neuronal activity and thus to establish synaptic connectivity and behavioral causality. Inhibitory channels are particularly advantageous to explore signal processing in neural circuits since they permit the functional removal of selected neurons on a trial-by-trial basis. However, applying these tools to study the visual system poses a considerable challenge because the illumination required for their activation usually also stimulates photoreceptors substantially, precluding the simultaneous probing of visual responses. Here, we explore the utility of the recently discovered anion channelrhodopsins GtACR1 and GtACR2 for application in the visual system of Drosophila. We first characterized their properties using a larval crawling assay. We further obtained whole-cell recordings from cells expressing GtACR1, which mediated strong and light-sensitive photocurrents. Finally, using physiological recordings and a behavioral readout, we demonstrate that GtACR1 enables the fast and reversible silencing of genetically targeted neurons within circuits engaged in visual processing.

Images projected onto the retina of an animal eye are rarely still. Instead, they usually contain motion signals originating either from moving objects or from retinal slip caused by self-motion. Accordingly, motion signals tell the animal in which direction a predator, prey, or the animal itself is moving. At the neural level, visual motion detection has been proposed to extract directional information by a delay-and-compare mechanism, representing a classic example of neural computation. Neurons responding selectively to motion in one but not in the other direction have been identified in many systems, most prominently in the mammalian retina and the fly optic lobe. Technological advances have now allowed researchers to characterize these neurons’ upstream circuits in exquisite detail. Focusing on these upstream circuits, we review and compare recent progress in understanding the mechanisms that generate direction selectivity in the early visual system of mammals and flies.

Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.

Visual perception seems effortless to us, yet it is the product of elaborate signal processing in intricate brain circuits. Apart from vertebrates, arthropods represent another major animal group with sophisticated visual systems in which the underlying mechanisms can be studied. Arthropods feature identified neurons and other experimental advantages, facilitating an understanding of circuit function at the level of individual neurons and their synaptic interactions. Here, focusing on insect and crustacean species, we summarize and connect our current knowledge in four related areas of research: (1) elementary motion detection in early visual processing; (2) the detection of higher level visual features such as optic flow fields, small target motion and object distance; (3) the integration of such signals with other sensory modalities; and (4) state-dependent visual motion processing.

Understanding how the brain controls behaviour is undisputedly one of the grand goals of neuroscience research, and the pursuit of this goal has a long tradition in insect neuroscience. However, appropriate techniques were lacking for a long time. Recent advances in genetic and recording techniques now allow the participation of identified neurons in the execution of specific behaviours to be interrogated. By focusing on fly visual course control, I highlight what has been learned about the neuronal circuit modules that control visual guidance in Drosophila melanogaster through the use of these techniques.

Dendritic integration is a fundamental element of neuronal information processing. So far, few studies have provided a detailed spatial picture of this process, describing the properties of local dendritic activity and its subcellular organization. Here, we used 2-photon calcium imaging in optic flow processing neurons of the fly Calliphora vicina to determine the preferred location and direction of local motion cues for small branchlets throughout the entire dendrite. We found a pronounced retinotopic mapping on both the subcellular and the cell population level. In addition, dendritic branchlets residing in different layers of the neuropil were tuned to distinct directions of motion. Summing the local receptive fields of all dendritic branchlets reproduced the characteristic properties of these neurons' axonal output receptive fields. Our results corroborate the notion that the dendritic morphology of vertical system cells allows them to selectively collect local motion inputs with particular directional preferences from a spatially organized input repertoire, thus forming filters that match global patterns of optic flow. Furthermore, we suggest that the facet arrangement across the fly's eye shapes the subcellular direction tuning to local motion stimuli. These data illustrate a highly structured circuit organization as an efficient way to hard-wire a complex sensory task.

Detecting the direction of image motion is a fundamental component of visual computation and is essential for survival of the animal. However, at the level of the photoreceptors, the direction in which the image is shifting locally is not explicitly represented. Rather, directional motion information needs to be extracted from the photoreceptor array by comparing the signals of neighboring photoreceptors over time. The exact nature of this process as implemented in the Drosophila visual system is currently being studied in great detail, and much progress has recently been made in determining the neural circuits giving rise to directional motion information in this species.

Visual systems extract directional motion information from spatiotemporal luminance changes on the retina. An algorithmic model, the Reichardt detector, accounts for this by multiplying adjacent inputs after asymmetric temporal filtering. The outputs of two mirror-symmetrical units tuned to opposite directions are thought to be subtracted on the dendrites of wide-field motion-sensitive lobula plate tangential cells by antagonistic transmitter systems. InDrosophila, small-field T4/T5 cells carry visual motion information to the tangential cells that are depolarized during preferred and hyperpolarized during null direction motion. While preferred direction input is likely provided by excitation from T4/T5 terminals, the origin of null direction inhibition is unclear. Probing the connectivity between T4/T5 and tangential cells inDrosophilausing a combination of optogenetics, electrophysiology, and pharmacology, we found a direct excitatory as well as an indirect inhibitory component. This suggests that the null direction response is caused by feedforward inhibition via yet unidentified neurons.

Approaches are sought after to regulate ionotropic and chronotropic properties of the mammalian heart. Electrodes are commonly used for rapidly exciting cardiac tissue and resetting abnormal pacing. With the advent of optogenetics and the use of tissue-specific expression of light-activated channels, cardiac cells cannot only be excited but also inhibited with ion-selective conductance. As a proof of concept for the ability to slow down cardiac pacing, anion-conducting channelrhodopsins (GtACR1/2) and the anion pump halorhodopsin (eNpHR) were expressed in hearts of larval Drosophila and activated by light. Unlike body wall muscles in most animals, the equilibrium potential for Cl&minus, is more positive as compared to the resting membrane potential in larval Drosophila. As a consequence, upon activating the two forms of GtACR1 and 2 with low light intensity the heart rate increased, likely due to depolarization and opening of voltage-gated Ca2+ channels. However, with very intense light activation the heart rate ceases, which may be due to Cl&ndash, shunting to the reversal potential for chloride. Activating eNpHR hyperpolarizes body wall and cardiac muscle in larval Drosophila and rapidly decreases heart rate. The decrease in heart rate is related to light intensity. Intense light activation of eNpHR stops the heart from beating, whereas lower intensities slowed the rate. Even with upregulation of the heart rate with serotonin, the pacing of the heart was slowed with light. Thus, regulation of the heart rate in Drosophila can be accomplished by activating anion-conducting channelrhodopsins using light. These approaches are demonstrated in a genetically amenable insect model.

Seeing the direction of motion is fundamental for visual navigation, predator avoidance, and prey capture. However, the direction of motion is not explicitly represented at the level of individual photoreceptors. Rather, directional motion information needs to be extracted from the photoreceptor array by comparing the signals of neighboring photoreceptors over time. The exact nature of this process as implemented in the Drosophila visual system is currently being studied in great detail, and much progress has recently been made in determining the neural circuits giving rise to directional motion information in this species.

When confronted with a large-field stimulus rotating around the vertical body axis, flies display a following behavior called “optomotor response.” As neural control elements, the large tangential horizontal system (HS) cells of the lobula plate have been prime candidates for long. Here, we applied optogenetic stimulation of HS cells to evaluate their behavioral role inDrosophila. To minimize interference of the optical activation of channelrhodopsin-2 with the visual perception of the flies, we used a bistable variant called ChR2-C128S. By applying pulses of blue and yellow light, we first demonstrate electrophysiologically that lobula plate tangential cells can be activated and deactivated repeatedly with no evident change in depolarization strength over trials. We next show that selective optogenetic activation of HS cells elicits robust yaw head movements and yaw turning responses in fixed and tethered flying flies, respectively.

The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. We circumvent this problem by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5 (ref. 2). Here we find that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours.

Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.

Detecting the direction of motion contained in the visual scene is crucial for many behaviors. However, because single photoreceptors only signal local luminance changes, motion detection requires a comparison of signals from neighboring photoreceptors across time in downstream neuronal circuits. For signals to coincide on readout neurons that thus become motion and direction selective, different input lines need to be delayed with respect to each other. Classical models of motion detection rely on non-linear interactions between two inputs after different temporal filtering. However, recent studies have suggested the requirement for at least three, not only two, input signals. Here, we comprehensively characterize the spatiotemporal response properties of all columnar input elements to the elementary motion detectors in the fruit fly, T4 and T5 cells, via two-photon calcium imaging. Between these input neurons, we find large differences in temporal dynamics. Based on this, computer simulations show that only a small subset of possible arrangements of these input elements maps onto a recently proposed algorithmic three-input model in a way that generates a highly direction-selective motion detector, suggesting plausible network architectures. Moreover, modulating the motion detection system by octopamine-receptor activation, we find the temporal tuning of T4 and T5 cells to be shifted toward higher frequencies, and this shift can be fully explained by the concomitant speeding of the input elements.

Drosophila has emerged as an important model organism for the study of the neural basis of behavior. Its main asset is the experimental accessibility of identified neurons by genetic manipulation and physiological recordings. Drosophila therefore offers the opportunity to reach an integrative understanding of the development and neural underpinnings of behavior at all processing stages, from sensing to motor control, in a single species. Here, we will provide an account of the procedures involved in recording the electrical potential of individual neurons in the visual system of adult Drosophila using the whole-cell patch-clamp method. To this end, animals are fixed to a holder and mounted below a recording chamber. The head capsule is cut open and the glial sheath covering the brain is ruptured by a combination of shearing and enzymatic digest. Neuronal somata are thus exposed and targeted by low-resistance patch electrodes. After formation of a high resistance seal, electrical access to the cell is gained by small current pulses and suction. Stable recordings of large neurons are feasible for1 h and can be combined with controlled visual stimulation as well as genetic and pharmacological manipulation of upstream circuit elements to infer circuit function in great detail.

The optic lobe of Drosophila houses about 60,000 neurons that are organized in parallel, retinotopically arranged columns. Based on the Golgi-staining method, Fischbach and Dittrich ([1989] Cell Tissue Res 258:441–475) determined that each column contains about 90 identified cells. Each of these cells is supposed to release one or two different neurotransmitters. However, for most cells the released neurotransmitter is not known. Here we characterize the vast majority of the neurons in the Drosophila optic lobe that release acetylcholine (Ach), the major excitatory neurotransmitter of the insect central nervous system. We employed a promoter specific for cholinergic neurons and restricted its activity to single or a few cells using the MARCM technique. This approach allowed us to establish an anatomical map of neurons with a cholinergic phenotype based on their branching pattern. We identified 43 different types of neurons with a cholinergic phenotype. Thirty-one of them match previously described members of nine different subgroups: Transmedullary (Tm), Transmedullary Y (TmY), Medulla intrinsic (Mi, Mt, and Pm), Bushy T (T), Translobula Plate (Tlp), and Lobula intrinsic (Lcn and Lt) neurons (Fischbach and Dittrich [1989]). Intriguingly, 12 newly identified cell types suggest that previous Golgi studies were not saturating and that the actual number of different neurons per column is higher than previously thought. This study and similar ones on other neurotransmitter systems will contribute towards a columnar wiring diagram and foster the functional dissection of the visual circuitry in Drosophila. J. Comp. Neurol. 519:162-176, 2011. © 2010 Wiley-Liss, Inc.

Zusammenfassung Visuelle, auf dem optischen Fluss basierende Kurskontrolle wurde sowohl an stationären als auch an frei fliegenden Fliegen ausgiebig studiert. Für die neuronale Kontrolle dieses Verhaltens scheinen die Tangentialzellen der Lobulaplatte eine große Rolle zu spielen. Diese Zellen sind bewegungsempfindlich, haben große rezeptive Felder, und die räumliche Verteilung ihrer Vorzugsrichtungen entspricht dem optischen Fluss, wie er bei bestimmten Flugmanövern auftritt. Der präsynaptische Schaltkreis, der die lokalen Bewegungsrichtungen berechnet und die Tangentialzellen überhaupt erst empfindlich für visuelle Bewegung macht, lag jedoch lange Zeit aufgrund der geringen Größe der entsprechenden Neurone im Dunkeln. Erste Erfolge erbrachte hier die Kombination von elektrophysiologischen Ableitungen der Tangentialzellen bei Drosophila mit genetischer Blockade einzelner Kandidaten-Neurone. Mit diesem Ansatz wurden die Lamina-Neurone L1 und L2 als Eingangselemente zu zwei parallelen Bewegungsdetektionssystemen identifiziert, die selektiv auf die Bewegung von Dunkel-Hell (L1, ON-System) bzw. Hell-Dunkel (L2, Off-System) -Übergängen reagieren.

Motion detection in the fly is extremely fast with low computational requirements. Inspired from the fly's vision system, we focus on a real-time flight control on a miniquadrotor with fast visual feedback. In this work, an elaborated elementary motion detector (EMD) is utilized to detect local optical flow. Combined with novel receptive field templates, the yaw rate of the quadrotor is estimated through a lookup table established with this bioinspired visual sensor. A closed-loop control system with the feedback of yaw rate estimated by EMD is designed. With the motion of the other degrees of freedom stabilized by a camera tracking system, the yaw-rate of the quadrotor during hovering is controlled based on EMD feedback under real-world scenario. The control performance of the proposed approach is compared with that of conventional approach. The experimental results demonstrate the effectiveness of utilizing EMD for quadrotor control.

Typically, neurons in sensory areas are highly interconnected. Coupling two neurons can synchronize their activity and affect a variety of single-cell properties, such as their stimulus tuning, firing rate or gain. All of these factors must be considered to understand how two neurons should be coupled to optimally process stimuli. We quantified the functional effect of an interaction between two optic-flow processing neurons (Vi and H1) in the fly (Lucilia sericata). Using a generative model, we estimated a uni-directional coupling from H1 to Vi. Especially at a low signal-to-noise ratio (SNR), the coupling strongly improved the information about optic-flow in Vi. We identified two constraints confining the strength of the interaction. First, for weak couplings, Vi benefited from inputs by H1 without a concomitant shift of its stimulus tuning. Second, at both low and high SNR, the coupling strength lay in a range in which the information carried by single spikes is optimal.

Motion vision provides essential cues for navigation and course control as well as for mate, prey, or predator detection. Consequently, neurons responding to visual motion in a direction-selective way are found in almost all species that see. However, directional information is not explicitly encoded at the level of a single photoreceptor. Rather, it has to be computed from the spatio-temporal excitation level of at least two photoreceptors. How this computation is done and how this computation is implemented in terms of neural circuitry and membrane biophysics have remained the focus of intense research over many decades. Here, we review recent progress made in this area with an emphasis on insects and the vertebrate retina.

SummaryRecent experiments have shown that motion detection in Drosophila starts with splitting the visual input into two parallel channels encoding brightness increments (ON) or decrements (OFF). This suggests the existence of either two (ON-ON, OFF-OFF) or four (for all pairwise interactions) separate motion detectors. To decide between these possibilities, we stimulated flies using sequences of ON and OFF brightness pulses while recording from motion-sensitive tangential cells. We found direction-selective responses to sequences of same sign (ON-ON, OFF-OFF), but not of opposite sign (ON-OFF, OFF-ON), refuting the existence of four separate detectors. Based on further measurements, we propose a model that reproduces a variety of additional experimental data sets, including ones that were previously interpreted as support for four separate detectors. Our experiments and the derived model mark an important step in guiding further dissection of the fly motion detection circuit.

Summary Moving animals experience constant sensory feedback, such as panoramic image shifts on the retina, termed optic flow. Underlying neuronal signals are thought to be important for exploratory behavior by signaling unintended course deviations and by providing spatial information about the environment [ 1 , 2 ]. Particularly in insects, the encoding of self-motion-related optic flow is well understood [ 1 , 2 , 3 , 4 , 5 ]. However, a gap remains in understanding how the associated neuronal activity controls locomotor trajectories. In flies, visual projection neurons belonging to two groups encode panoramic horizontal motion: horizontal system (HS) cells respond with depolarization to front-to-back motion and hyperpolarization to the opposite direction [ 6 , 7 ], and other neurons have the mirror-symmetrical response profile [ 6 , 8 , 9 ]. With primarily monocular sensitivity, the neurons’ responses are ambiguous for different rotational and translational self-movement components. Such ambiguities can be greatly reduced by combining signals from both eyes [ 10 , 11 , 12 ] to determine turning and movement speed [ 13 , 14 , 15 , 16 ]. Here, we explore the underlying functional logic by optogenetic HS cell manipulation in tethered walking Drosophila. We show that de- and hyperpolarization evoke opposite turning behavior, indicating that both direction-selective signals are transmitted to descending pathways for course control. Further experiments reveal a negative effect of bilaterally symmetric de- and hyperpolarization on walking velocity. Our results are therefore consistent with a functional architecture in which the HS cells’ membrane potential influences walking behavior bi-directionally via two decelerating pathways.

Motion vision is a major function of all visual systems, yet the underlying neural mechanisms and circuits are still elusive. In the lamina, the first optic neuropile of Drosophila melanogaster, photoreceptor signals split into five parallel pathways, L1-L5. Here we examine how these pathways contribute to visual motion detection by combining genetic block and reconstitution of neural activity in different lamina cell types with whole-cell recordings from downstream motion-sensitive neurons. We find reduced responses to moving gratings if L1 or L2 is blocked; however, reconstitution of photoreceptor input to only L1 or L2 results in wild-type responses. Thus, the first experiment indicates the necessity of both pathways, whereas the second indicates sufficiency of each single pathway. This contradiction can be explained by electrical coupling between L1 and L2, allowing for activation of both pathways even when only one of them receives photoreceptor input. A fundamental difference between the L1 pathway and the L2 pathway is uncovered when blocking L1 or L2 output while presenting moving edges of positive (ON) or negative (OFF) contrast polarity: blocking L1 eliminates the response to moving ON edges, whereas blocking L2 eliminates the response to moving OFF edges. Thus, similar to the segregation of photoreceptor signals in ON and OFF bipolar cell pathways in the vertebrate retina, photoreceptor signals segregate into ON-L1 and OFF-L2 channels in the lamina of Drosophila.

SUMMARY A central goal in sensory neuroscience is to fully characterize a neuron’s input-output relation. However, strong nonlinearities in the responses of sensory neurons have made it difficult to develop models that generalize to arbitrary stimuli. Typically, the standard linear-nonlinear models break down when neurons exhibit stimulus-dependent modulations of their gain or selectivity. We studied these issues in optic-flow processing neurons in the fly. We found that the neurons’ receptive fields are fully described by a time-varying vector field that is space-time separable. Increasing the stimulus strength, however, strongly reduces the neurons’ gain and selectivity. To capture these changes in response behavior, we extended the linear-nonlinear model by a biophysically motivated gain and selectivity mechanism. We fit all model parameters directly to the data and show that the model now characterizes the neurons’ input-output relation well over the full range of motion stimuli.