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.

A common directional tuning mechanism of Drosophila motion-sensing neurons in the ON and in the OFF pathway.

In the fruit fly optic lobe, T4 and T5 cells represent the first direction-selective neurons, with T4 cells responding selectively to moving brightness increments (ON) and T5 cells to brightness decrements (OFF). Both T4 and T5 cells comprise four subtypes with directional tuning to one of the four cardinal directions. We had previously found that upward-sensitive T4 cells implement both preferred direction enhancement and null direction suppression (Haag et al., 2016). Here, we asked whether this mechanism generalizes to OFF-selective T5 cells and to all four subtypes of both cell classes. We found that all four subtypes of both T4 and T5 cells implement both mechanisms, that is preferred direction enhancement and null direction inhibition, on opposing sides of their receptive fields. This gives rise to the high degree of direction selectivity observed in both T4 and T5 cells within each subpopulation.

Complementary mechanisms create direction selectivity in the fly.

How neurons become sensitive to the direction of visual motion represents a classic example of neural computation. Two alternative mechanisms have been discussed in the literature so far: preferred direction enhancement, by which responses are amplified when stimuli move along the preferred direction of the cell, and null direction suppression, where one signal inhibits the response to the subsequent one when stimuli move along the opposite, i.e. null direction. Along the processing chain in the Drosophila optic lobe, directional responses first appear in T4 and T5 cells. Visually stimulating sequences of individual columns in the optic lobe with a telescope while recording from single T4 neurons, we find both mechanisms at work implemented in different sub-regions of the receptive field. This finding explains the high degree of directional selectivity found already in the fly's primary motion-sensing neurons and marks an important step in our understanding of elementary motion detection.

Subcellular mapping of dendritic activity in optic flow processing neurons.

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.

A directional tuning map of Drosophila elementary motion detectors.

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.

Dendritic end inhibition in large-field visual neurons of the fly.

The extraction of optic flow fields by visual systems is crucial for course stabilization during locomotion, and relies on feedforward and lateral integration of visual inputs. Here we report a novel form of systemic, motion-sensitive lateral suppression in the dendrites of large, flow-field-selective neurons in the fly's visual lobes. Using in vivo Calcium-imaging and intracellular recordings, we demonstrate that responses in dendrites, but not axon terminals, are end inhibited by flanking gratings both in the vertical and horizontal systems. We show evidence for a mechanism involving wide-field dendritic inhibition that exceeds the retinotopic spatial extent of the dendrites. Using compartmental modeling, we point out a possible function in enhancing selectivity for optic flow fields. Our results suggest that lateral suppression is a common element serving similar functional requirements in different visual systems.

Flight activity alters velocity tuning of fly motion-sensitive neurons.

Sensory neurons are mostly studied in fixed animals, but their response properties might change when the animal is free to move. Indeed, recent studies found differences between responses of sensory neurons in resting versus moving insects. Since the dynamic range of visual motion stimuli strongly depends on the speed at which an animal is moving, we investigated whether the visual system adapts to such changes in stimulus dynamics as induced by self-motion. Lobula plate tangential cells of flies lend themselves well to study this question because they are known to code for ego-motion based on optic-flow. We recorded the responses of the lobula plate tangential cell H1 to a visual pattern moving at different velocities under three different conditions: fixed flies before and after application of the octopamine agonist chlordimeform (CDM) and tethered flying flies. CDM has been previously shown to induce arousal in flies. We found that flying as well as the application of CDM significantly broadens the velocity tuning of H1 toward higher velocities.

Central gating of fly optomotor response.

We study the integration of multisensory and central input at the level of an identified fly motoneuron, the ventral cervical nerve motoneuron (VCNM) cell, which controls head movements of the animal. We show that this neuron receives input from a central neuron signaling flight activity, from two identified wide-field motion-sensitive neurons, from the wind-sensitive Johnston organ on the antennae, and from the campaniform sensillae of the halteres. We find that visual motion alone leads to only subthreshold responses. Only when it is combined with flight activity or wind stimuli does the VCNM respond to visual motion by modulating its spike activity in a directionally selective way. This nonlinear enhancement of visual responsiveness in the VCNM by central activity is reflected at the behavioral level, when compensatory head movements are measured in response to visual motion. While head movements of flies have only a small amplitude when flies are at rest, the response amplitude is increased by a factor of 30-40 during flight.

Fly motion vision.

Fly motion vision and resultant compensatory optomotor responses are a classic example for neural computation. Here we review our current understanding of processing of optic flow as generated by an animal's self-motion. Optic flow processing is accomplished in a series of steps: First, the time-varying photoreceptor signals are fed into a two-dimensional array of Reichardt-type elementary motion detectors (EMDs). EMDs compute, in parallel, local motion vectors at each sampling point in space. Second, the output signals of many EMDs are spatially integrated on the dendrites of large-field tangential cells in the lobula plate. In the third step, tangential cells form extensive interactions with each other, giving rise to their large and complex receptive fields. Thus, tangential cells can act as matched filters tuned to optic flow during particular flight maneuvers. They finally distribute their information onto postsynaptic descending neurons, which either instruct the motor centers of the thoracic ganglion for flight and locomotion control or act themselves as motor neurons that control neck muscles for head movements.

Robust coding of ego-motion in descending neurons of the fly.

In many species, motion-sensitive neurons responding to optic flow at higher processing stages are well characterized; however, less is known how this representation of ego-motion is further transformed into an appropriate motor response. Here, we analyzed in the blowfly Calliphora vicina the visuomotor transformation from motion-sensitive neurons in the lobula plate [V2 and vertical system (VS) cells] onto premotor descending neurons [descending neurons of the ocellar and vertical system (DNOVS) cells] feeding into the motor circuit of the fly thoracic ganglion. We found that each of these cells is tuned to rotation of the fly around a particular body axis. Comparing the responses of presynaptic and postsynaptic cells revealed that DNOVS cells have approximately the same tuning widths as V2 and VS cells. However, DNOVS signals cells are less corrupted by fluctuations arising from the spatial structure of the visual input than their presynaptic elements. This leads to a more robust representation of ego-motion at the level of descending neurons. Thus, when moving from lobula plate cells to descending neurons, the selectivity for a particular optic flow remains unaltered, but the robustness of the representation increases.

Local and global motion preferences in descending neurons of the fly.

For a moving animal, optic flow is an important source of information about its ego-motion. In flies, the processing of optic flow is performed by motion sensitive tangential cells in the lobula plate. Amongst them, cells of the vertical system (VS cells) have receptive fields with similarities to optic flows generated during rotations around different body axes. Their output signals are further processed by pre-motor descending neurons. Here, we investigate the local motion preferences of two descending neurons called descending neurons of the ocellar and vertical system (DNOVS1 and DNOVS2). Using an LED arena subtending 240 degrees x 95 degrees of visual space, we mapped the receptive fields of DNOVS1 and DNOVS2 as well as those of their presynaptic elements, i.e. VS cells 1-10 and V2. The receptive field of DNOVS1 can be predicted in detail from the receptive fields of those VS cells that are most strongly coupled to the cell. The receptive field of DNOVS2 is a combination of V2 and VS cells receptive fields. Predicting the global motion preferences from the receptive field revealed a linear spatial integration in DNOVS1 and a superlinear spatial integration in DNOVS2. In addition, the superlinear integration of V2 output is necessary for DNOVS2 to differentiate between a roll rotation and a lift translation of the fly.

Different receptive fields in axons and dendrites underlie robust coding in motion-sensitive neurons.

In the visual system of the blowfly Calliphora vicina, neurons of the vertical system (VS cells) integrate wide-field motion information from a retinotopic array of local motion detectors. In vivo calcium imaging reveals two distinct and separate receptive fields in these cells: a narrow dendritic receptive field corresponding to feedforward input from the local motion detectors and a broad axon terminal receptive field that additionally incorporates input from neighboring cells mediated by lateral axo-axonal gap junctions. We show that the axon terminal responses are linear interpolations of the dendritic responses, resulting in a robust population coding of optic flow parameters as predicted by previous modeling studies. Compartmental modeling shows that spatially separating the axonal gap junctions from the conductive load of the dendritic synapses increases the coupling strength of the gap junctions, making this interpolation possible.

The morphological identity of insect dendrites.

Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.

Nonlinear integration of binocular optic flow by DNOVS2, a descending neuron of the fly.

For visual orientation and course stabilization, flies rely heavily on the optic flow perceived by the animal during flight. The processing of optic flow is performed in motion-sensitive tangential cells of the lobula plate, which are well described with respect to their visual response properties and the connectivity among them. However, little is known about the postsynaptic descending neurons, which convey motion information to the motor circuits in the thoracic ganglion. Here we investigate the physiology and connectivity of an identified premotor descending neuron, called DNOVS2 (for descending neuron of the ocellar and vertical system). We find that DNOVS2 is tuned in a supralinear way to rotation around the longitudinal body axis. Experiments involving stimulation of the ipsilateral and the contralateral eye indicate that ipsilateral computation of motion information is modified nonlinearly by motion information from the contralateral eye. Performing double recordings of DNOVS2 and lobula plate tangential cells, we find that DNOVS2 is connected ipsilaterally to a subset of vertical-sensitive cells. From the contralateral eye, DNOVS2 receives input most likely from V2, a heterolateral spiking neuron. This specific neural circuit is sufficient for the tuning of DNOVS2, making it probably an important element in optomotor roll movements of the head and body around the fly's longitudinal axis.

Electrical coupling of lobula plate tangential cells to a heterolateral motion-sensitive neuron in the fly.

Many motion-sensitive tangential cells of the lobula plate in blowflies are well described with respect to their visual response properties and the connectivity among them. In addition to extensive connections between tangential cells within the lobula plate of one brain hemisphere, there exist many connections between the two hemispheres. Most of these connections have been found for neurons sensitive to horizontal motion. For neurons sensitive to vertical motion, however, only the connection of vertical sensitive cells (VS cells) and a cell (V1 cell) projecting to the other hemisphere has been demonstrated thus far. The ability to identify the presynaptic and postsynaptic cells as well as the good accessibility has made this specific synapse a model for graded transmission of synapses. However, the exact type of synapse, electrical or chemical, is not known. Investigating the connectivity between VS cells 1-3 and the V1 cell by means of dual recordings, we find that the VS cells are coupled via electrical synapses to the V1 cell. The results were confirmed by visualizing dye coupling between VS cells and V1.

Robust coding of flow-field parameters by axo-axonal gap junctions between fly visual interneurons.

Complex flight maneuvers require a sophisticated system to exploit the optic flow resulting from moving images of the environment projected onto the retina. In the fly's visual course control center, the lobula plate, 10 so-called vertical system (VS) cells are thought to match, with their complex receptive fields, the optic flow resulting from rotation around different body axes. However, signals of single VS cells are unreliable indicators of such optic flow parameters in the context of their noisy, texture-dependent input from local motion measurements. Here we propose an alternative encoding scheme based on network simulations of biophysically realistic compartmental models of VS cells. The simulations incorporate recent data about the highly selective connectivity between VS cells consisting of an electrical axo-axonal coupling between adjacent cells and a reciprocal inhibition between the most distant cells. We find that this particular wiring performs a linear interpolation between the output signals of VS cells, leading to a robust representation of the axis of rotation even in the presence of textureless patches of the visual surround.

Integration of lobula plate output signals by DNOVS1, an identified premotor descending neuron.

Many motion-sensitive tangential cells of the lobula plate in blowflies are well described with respect to their visual response properties and the connectivity among them. They have large and complex receptive fields with different preferred directions in different parts of their receptive fields matching the optic flow that occurs during various flight maneuvers. However, much less is known about how tangential cells connect to postsynaptic neurons descending to the motor circuits in the thoracic ganglion and how optic flow is represented in these downstream neurons. Here we describe the physiology and the connectivity of a prominent descending neuron called DNOVS1 (for descending neurons of the ocellar and vertical system). We find that DNOVS1 is electrically coupled to a subset of vertical system cells. The specific wiring leads to a preference of DNOVS1 for rotational flow fields around a particular body axis. In addition, DNOVS1 receives input from interneurons connected to the ocelli.

Reciprocal inhibitory connections within a neural network for rotational optic-flow processing.

Neurons in the visual system of the blowfly have large receptive fields that are selective for specific optic flow fields. Here, we studied the neural mechanisms underlying flow-field selectivity in proximal Vertical System (VS)-cells, a particular subset of tangential cells in the fly. These cells have local preferred directions that are distributed such as to match the flow field occurring during a rotation of the fly. However, the neural circuitry leading to this selectivity is not fully understood. Through dual intracellular recordings from proximal VS cells and other tangential cells, we characterized the specific wiring between VS cells themselves and between proximal VS cells and horizontal sensitive tangential cells. We discovered a spiking neuron (Vi) involved in this circuitry that has not been described before. This neuron turned out to be connected to proximal VS cells via gap junctions and, in addition, it was found to be inhibitory onto VS1.

Nonlinear, binocular interactions underlying flow field selectivity of a motion-sensitive neuron.

Neurons in many species have large receptive fields that are selective for specific optic flow fields. Here, we studied the neural mechanisms underlying flow field selectivity in lobula plate tangential cells (LPTCs) of the blowfly. Among these cells, the H2 cell responds preferentially to visual stimuli approximating rotational optic flow. Through double recordings from H2 and many other LPTCs, we characterized a bidirectional commissural pathway that allows visual information to be shared between the hemispheres. This pathway is mediated by axo-axonal electrical coupling of H2 and the horizontal system equatorial (HSE) cell located in the opposite hemisphere. Using single-cell ablations, we found that this pathway is sufficient to allow H2 to amplify and attenuate dendritic input during binocular visual stimuli. This is accomplished through a modulation of H2's membrane potential by input from the contralateral HSE cell, which scales the firing rate of H2 during visual stimulation but is not sufficient to induce action potentials.

Sharing receptive fields with your neighbors: tuning the vertical system cells to wide field motion.

In the blowfly, the direction-selective response of the 60 lobula-plate tangential cells has been ascribed to the integration of local motion information across their extensive dendritic trees. Because the lobula plate is organized retinotopically, the receptive fields of the tangential cells ought to be determined by their dendritic architecture. However, this appears not always to be the case. One compelling example is the exceptionally wide receptive fields of the vertical system (VS) tangential cells. Using dual-intracellular recordings, Haag and Borst (2004) found VS cells to be mutually coupled in such a way that each VS cell is connected exclusively to its immediate neighbors. This coupling may form the basis of the broad receptive fields of VS cells. Here, we tested this hypothesis directly by photoablating individual VS cells. The receptive field width of VS cells indeed narrowed after the ablation of single VS cells, specifically depending on whether the receptive field of the ablated cell was more frontal or more posterior to the recorded cell. In particular, the responses changed as if the neuron lost access to visual information from the ablated neuron and those VS cells more distal than it from the recorded neuron. These experiments provide strong evidence that the lateral connections among VS cells are a crucial component in the mechanism underlying their complex receptive fields, augmenting the direct columnar input to their dendrites.