An open platform for visual stimulation of insects.

To study how the nervous system processes visual information, experimenters must record neural activity while delivering visual stimuli in a controlled fashion. In animals with a nearly panoramic field of view, such as flies, precise stimulation of the entire visual field is challenging. We describe a projector-based device for stimulation of the insect visual system under a microscope. The device is based on a bowl-shaped screen that provides a wide and nearly distortion-free field of view. It is compact, cheap, easy to assemble, and easy to operate using the included open-source software for stimulus generation. We validate the virtual reality system technically and demonstrate its capabilities in a series of experiments at two levels: the cellular, by measuring the membrane potential responses of visual interneurons; and the organismal, by recording optomotor and fixation behavior of Drosophila melanogaster in tethered flight. Our experiments reveal the importance of stimulating the visual system of an insect with a wide field of view, and we provide a simple solution to do so.

Connectivity Matrix Seriation via Relaxation.

Volume electron microscopy together with computer-based image analysis are yielding neural circuit diagrams of ever larger regions of the brain. These datasets are usually represented in a cell-to-cell connectivity matrix and contain important information about prevalent circuit motifs allowing to directly test various theories on the computation in that brain structure. Of particular interest are the detection of cell assemblies and the quantification of feedback, which can profoundly change circuit properties. While the ordering of cells along the rows and columns doesn't change the connectivity, it can make special connectivity patterns recognizable. For example, ordering the cells along the flow of information, feedback and feedforward connections are segregated above and below the main matrix diagonal, respectively. Different algorithms are used to renumber matrices such as to minimize a given cost function, but either their performance becomes unsatisfying at a given size of the circuit or the CPU time needed to compute them scales in an unfavorable way with increasing number of neurons. Based on previous ideas, I describe an algorithm which is effective in matrix reordering with respect to both its performance as well as to its scaling in computing time. Rather than trying to reorder the matrix in discrete steps, the algorithm transiently relaxes the integer program by assigning a real-valued parameter to each cell describing its location on a continuous axis ('smooth-index') and finds the parameter set that minimizes the cost. I find that the smooth-index algorithm outperforms all algorithms I compared it to, including those based on topological sorting.

Multilevel visual motion opponency in Drosophila.

Inhibitory interactions between opponent neuronal pathways constitute a common circuit motif across brain areas and species. However, in most cases, synaptic wiring and biophysical, cellular and network mechanisms generating opponency are unknown. Here, we combine optogenetics, voltage and calcium imaging, connectomics, electrophysiology and modeling to reveal multilevel opponent inhibition in the fly visual system. We uncover a circuit architecture in which a single cell type implements direction-selective, motion-opponent inhibition at all three network levels. This inhibition, mediated by GluClα receptors, is balanced with excitation in strength, despite tenfold fewer synapses. The different opponent network levels constitute a nested, hierarchical structure operating at increasing spatiotemporal scales. Electrophysiology and modeling suggest that distributing this computation over consecutive network levels counteracts a reduction in gain, which would result from integrating large opposing conductances at a single instance. We propose that this neural architecture provides resilience to noise while enabling high selectivity for relevant sensory information.

How Flies See Motion.

How neurons detect the direction of motion is a prime example of neural computation: Motion vision is found in the visual systems of virtually all sighted animals, it is important for survival, and it requires interesting computations with well-defined linear and nonlinear processing steps-yet the whole process is of moderate complexity. The genetic methods available in the fruit fly Drosophila and the charting of a connectome of its visual system have led to rapid progress and unprecedented detail in our understanding of how neurons compute the direction of motion in this organism. The picture that emerged incorporates not only the identity, morphology, and synaptic connectivity of each neuron involved but also its neurotransmitters, its receptors, and their subcellular localization. Together with the neurons' membrane potential responses to visual stimulation, this information provides the basis for a biophysically realistic model of the circuit that computes the direction of visual motion.

Contrast normalization affects response time-course of visual interneurons.

In natural environments, light intensities and visual contrasts vary widely, yet neurons have a limited response range for encoding them. Neurons accomplish that by flexibly adjusting their dynamic range to the statistics of the environment via contrast normalization. The effect of contrast normalization is usually measured as a reduction of neural signal amplitudes, but whether it influences response dynamics is unknown. Here, we show that contrast normalization in visual interneurons of Drosophila melanogaster not only suppresses the amplitude but also alters the dynamics of responses when a dynamic surround is present. We present a simple model that qualitatively reproduces the simultaneous effect of the visual surround on the response amplitude and temporal dynamics by altering the cells' input resistance and, thus, their membrane time constant. In conclusion, single-cell filtering properties as derived from artificial stimulus protocols like white-noise stimulation cannot be transferred one-to-one to predict responses under natural conditions.

Connecting Connectomes to Physiology.

With the advent of volumetric EM techniques, large connectomic datasets are being created, providing neuroscience researchers with knowledge about the full connectivity of neural circuits under study. This allows for numerical simulation of detailed, biophysical models of each neuron participating in the circuit. However, these models typically include a large number of parameters, and insight into which of these are essential for circuit function is not readily obtained. Here, we review two mathematical strategies for gaining insight into connectomics data: linear dynamical systems analysis and matrix reordering techniques. Such analytical treatment can allow us to make predictions about time constants of information processing and functional subunits in large networks.SIGNIFICANCE STATEMENT This viewpoint provides a concise overview on how to extract important insights from Connectomics data by mathematical methods. First, it explains how new dynamics and new time constants can evolve, simply through connectivity between neurons. These new time-constants can be far longer than the intrinsic membrane time-constants of the individual neurons. Second, it summarizes how structural motifs in the circuit can be discovered. Specifically, there are tools to decide whether or not a circuit is strictly feed-forward or whether feed-back connections exist. Only by reordering connectivity matrices can such motifs be made visible.

Disynaptic inhibition shapes tuning of OFF-motion detectors in Drosophila.

The circuitry underlying the detection of visual motion in Drosophila melanogaster is one of the best studied networks in neuroscience. Lately, electron microscopy reconstructions, algorithmic models, and functional studies have proposed a common motif for the cellular circuitry of an elementary motion detector based on both supralinear enhancement for preferred direction and sublinear suppression for null-direction motion. In T5 cells, however, all columnar input neurons (Tm1, Tm2, Tm4, and Tm9) are excitatory. So, how is null-direction suppression realized there? Using two-photon calcium imaging in combination with thermogenetics, optogenetics, apoptotics, and pharmacology, we discovered that it is via CT1, the GABAergic large-field amacrine cell, where the different processes have previously been shown to act in an electrically isolated way. Within each column, CT1 receives excitatory input from Tm9 and Tm1 and provides the sign-inverted, now inhibitory input signal onto T5. Ablating CT1 or knocking down GABA-receptor subunit Rdl significantly broadened the directional tuning of T5 cells. It thus appears that the signal of Tm1 and Tm9 is used both as an excitatory input for preferred direction enhancement and, through a sign inversion within the Tm1/Tm9-CT1 microcircuit, as an inhibitory input for null-direction suppression.

Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 Neurons.

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 female Drosophila 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 STATEMENT The 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 of Drosophila in 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.

Spatial and temporal control of expression with light-gated LOV-LexA.

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.

Anatomical distribution and functional roles of electrical synapses in Drosophila.

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.

A biophysical account of multiplication by a single neuron.

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.

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.

Maximally efficient prediction in the early fly visual system may support evasive flight maneuvers.

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.

Neural mechanism of spatio-chromatic opponency in the Drosophila amacrine neurons.

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.

Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons.

Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to our understanding of neural computation. Here, we developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: The flippase-dependent expression of GFP-tagged receptor subunits in single neurons and 'FlpTag', a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, we investigated the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system. We discovered a strictly segregated subcellular distribution of these proteins and a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors along the dendrite that matched the previously reported EM-reconstructed synapse distributions.

Seeing Natural Images through the Eye of a Fly with Remote Focusing Two-Photon Microscopy.

Visual systems of many animals, including the fruit fly Drosophila, represent the surrounding space as 2D maps, formed by populations of neurons. Advanced genetic tools make the fly visual system especially well accessible. However, in typical in vivo preparations for two-photon calcium imaging, relatively few neurons can be recorded at the same time. Here, we present an extension to a conventional two-photon microscope, based on remote focusing, which enables real-time rotation of the imaging plane, and thus flexible alignment to cellular structures, without resolution or speed trade-off. We simultaneously record from over 100 neighboring cells spanning the 2D retinotopic map. We characterize its representation of moving natural images, which we find is comparable to noise predictions. Our method increases throughput 10-fold and allows us to visualize a significant fraction of the fly's visual field. Furthermore, our system can be applied in general for a more flexible investigation of neural circuits.

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.

Dynamic Signal Compression for Robust Motion Vision in Flies.

Sensory systems need to reliably extract information from highly variable natural signals. Flies, for instance, use optic flow to guide their course and are remarkably adept at estimating image velocity regardless of image statistics. Current circuit models, however, cannot account for this robustness. Here, we demonstrate that the Drosophila visual system reduces input variability by rapidly adjusting its sensitivity to local contrast conditions. We exhaustively map functional properties of neurons in the motion detection circuit and find that local responses are compressed by surround contrast. The compressive signal is fast, integrates spatially, and derives from neural feedback. Training convolutional neural networks on estimating the velocity of natural stimuli shows that this dynamic signal compression can close the performance gap between model and organism. Overall, our work represents a comprehensive mechanistic account of how neural systems attain the robustness to carry out survival-critical tasks in challenging real-world environments.

How fly neurons compute the direction of visual motion.

Detecting the direction of image motion is a fundamental component of visual computation, essential for survival of the animal. However, at the level of individual photoreceptors, the direction in which the image is shifting is not explicitly represented. Rather, directional motion information needs to be extracted from the photoreceptor array by comparing the signals of neighboring units over time. The exact nature of this process as implemented in the visual system of the fruit fly Drosophila melanogaster has been studied in great detail, and much progress has recently been made in determining the neural circuits giving rise to directional motion information. The results reveal the following: (1) motion information is computed in parallel ON and OFF pathways. (2) Within each pathway, T4 (ON) and T5 (OFF) cells are the first neurons to represent the direction of motion. Four subtypes of T4 and T5 cells exist, each sensitive to one of the four cardinal directions. (3) The core process of direction selectivity as implemented on the dendrites of T4 and T5 cells comprises both an enhancement of signals for motion along their preferred direction as well as a suppression of signals for motion along the opposite direction. This combined strategy ensures a high degree of direction selectivity right at the first stage where the direction of motion is computed. (4) At the subsequent processing stage, tangential cells spatially integrate direct excitation from ON and OFF-selective T4 and T5 cells and indirect inhibition from bi-stratified LPi cells activated by neighboring T4/T5 terminals, thus generating flow-field-selective responses.

Optic flow-based course control in insects.

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.