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.

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.

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.

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.

The Effects of Chloride Flux on Drosophila Heart Rate.

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.

Bi-directional Control of Walking Behavior by Horizontal Optic Flow Sensors.

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 [1-5]. 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 [10-12] to determine turning and movement speed [13-16]. 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.

Optogenetic Neuronal Silencing in Drosophila during Visual Processing.

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.

Visual Circuits for Direction Selectivity.

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.

Electrophysiological Recordings from Lobula Plate Tangential Cells in Drosophila.

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 for >1 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.

Neural Circuit to Integrate Opposing Motions in the Visual Field.

When 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.

Development of connectivity in a motoneuronal network in Drosophila larvae.

BACKGROUND: Much of our understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. We know considerably less about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. Here, we investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. RESULTS: We find that as animals grow, motoneurons increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. CONCLUSIONS: In the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.

Optogenetic and pharmacologic dissection of feedforward inhibition in Drosophila motion vision.

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. In Drosophila, 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 in Drosophila using 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.

Optogenetic control of fly optomotor responses.

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 in Drosophila. 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.

Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila.

In recent years, Drosophila melanogaster has emerged as a powerful model for neuronal circuit development, pathology, and function. A major impediment to these studies has been the lack of a genetically encoded, specific, universal, and phenotypically neutral marker of the somatodendritic compartment. We have developed such a marker and show that it is effective and specific in all neuronal populations tested in the peripheral and central nervous system. The marker, which we name DenMark (Dendritic Marker), is a hybrid protein of the mouse protein ICAM5/Telencephalin and the red fluorescent protein mCherry. We show that DenMark is a powerful tool for revealing novel aspects of the neuroanatomy of developing dendrites, identifying previously unknown dendritic arbors, and elucidating neuronal connectivity.