Functional and developmental characterization of local motion sensing neurons in the fly visual system

Sighted animals use visual motion information to navigate in their environment, to search for food sources or mating partners and to avoid potentials predators. However, directional motion information is not explicitly represented in the photoreceptor signals, but rather needs to be extracted by postsynaptic circuits. For such a motion computation, different algorithmic models were proposed. The most prominent model multiplies the signal of two neighboring photoreceptors after one of them was temporally delayed. Fruit flies are well suited as a model organism to study the neuronal mechanisms underlying motion perception. With a low spatial but high temporal visual resolution, fruit flies are able to detect many different kinds of motion stimuli and perform a wide range of visually evoked behaviors. Thanks to the multitude of genetic tools optimized for Drosophila melanogaster, detailed manipulation of neuronal function can be performed on a molecular as well as on a cellular level. These tools allow to dissect the components of a neuronal circuit and investigate their respective function. In the visual system of flies exist neurons sensitive to wide field motion, which are important for the course control of flies. An open question remains the computation of upstream neurons detecting local motion. During my doctoral work I studied various aspects of the local motion sensing cells in the fly visual system: their functional properties, their importance for different behavioral tasks as well as their differentiation during development. In the first manuscript included in this thesis, we demonstrated that T4 and T5 cells are the elementary local motion sensing neurons of the fly. Calcium activity imaging of T4 and T5 cells revealed that four subtypes exist, each sensitive to motion along one of the four cardinal directions. Moreover, T4 cells responded specifically to light increments and T5 cells to light decrements. Blocking T4 neurons abolished the ON motion responses of postsynaptic lobula plate tangential cells. Accordingly, inactivating T5 cells inhibited the reaction of lobula plate tangential cells to OFF motion. We confirmed this effect by examining the turning behavior of walking flies with either T4 or T5 cells blocked. Flies without T4 output responded only to OFF edge motion, while flies with blocked T5 cells responded exclusively to ON edge motion. To investigate the functional role of the local motion sensing T4 and T5 cells, we studied the consequences of blocking these neurons and tested visual behavior. In the second manuscript, we described that inactivating T4 and T5 cells abolished the optomotor turning response of the flies. However, the motion blind flies were still able to orient towards a dark, vertical bar. Wedemonstrated that flies respond to the position of a bar independent of a motion cue. Therefore, we concluded that flies use positional as well as motion information to orient towards an attractive object. In the third manuscript, we further investigated the role of T4 and T5 cells in flight behavior and found these cells involved in the detection of expansion motion. Flight avoidance turns as well as landing responses of flies depend on functional T4 and T5 cells. These behaviors are evoked by expansion motion like a looming stimulus, which mimics an approaching predator or object. The importance of T4 and T5 cells for looming evoked behavior suggests, that these cells are not only connected to lobula plate tangential cells, which respond to rotatory wide-field motion, but are also presynaptic to looming sensitive neurons in the lobula plate. The last manuscript describes transcription factors important for the differentiation of T4 and T5 neurons. The morphology of all T4 and T5 subtypes is comparable; their dendrites are oriented opposite to the preferred direction of the cell and the axon terminals target one of the four lobula plate layers. Both the dendrites and the axon terminals are limited to only one layer of their respective neuropil. We found two postmitotic transcription factors expressed in the young T4 and T5 cells, SoxN and Sox102F, which regulate the common features of all subtypes. These transcription factors are crucial for the proper morphology of the T4 and T5 cells, as well as the function of the adult neurons

Functional and developmental characterization of local motion sensing neurons in the fly visual system

Sighted animals use visual motion information to navigate in their environment, to search for food sources or mating partners and to avoid potentials predators. However, directional motion information is not explicitly represented in the photoreceptor signals, but rather needs to be extracted by postsynaptic circuits. For such a motion computation, different algorithmic models were proposed. The most prominent model multiplies the signal of two neighboring photoreceptors after one of them was temporally delayed. Fruit flies are well suited as a model organism to study the neuronal mechanisms underlying motion perception. With a low spatial but high temporal visual resolution, fruit flies are able to detect many different kinds of motion stimuli and perform a wide range of visually evoked behaviors. Thanks to the multitude of genetic tools optimized for Drosophila melanogaster, detailed manipulation of neuronal function can be performed on a molecular as well as on a cellular level. These tools allow to dissect the components of a neuronal circuit and investigate their respective function. In the visual system of flies exist neurons sensitive to wide field motion, which are important for the course control of flies. An open question remains the computation of upstream neurons detecting local motion. During my doctoral work I studied various aspects of the local motion sensing cells in the fly visual system: their functional properties, their importance for different behavioral tasks as well as their differentiation during development. In the first manuscript included in this thesis, we demonstrated that T4 and T5 cells are the elementary local motion sensing neurons of the fly. Calcium activity imaging of T4 and T5 cells revealed that four subtypes exist, each sensitive to motion along one of the four cardinal directions. Moreover, T4 cells responded specifically to light increments and T5 cells to light decrements. Blocking T4 neurons abolished the ON motion responses of postsynaptic lobula plate tangential cells. Accordingly, inactivating T5 cells inhibited the reaction of lobula plate tangential cells to OFF motion. We confirmed this effect by examining the turning behavior of walking flies with either T4 or T5 cells blocked. Flies without T4 output responded only to OFF edge motion, while flies with blocked T5 cells responded exclusively to ON edge motion. To investigate the functional role of the local motion sensing T4 and T5 cells, we studied the consequences of blocking these neurons and tested visual behavior. In the second manuscript, we described that inactivating T4 and T5 cells abolished the optomotor turning response of the flies. However, the motion blind flies were still able to orient towards a dark, vertical bar. Wedemonstrated that flies respond to the position of a bar independent of a motion cue. Therefore, we concluded that flies use positional as well as motion information to orient towards an attractive object. In the third manuscript, we further investigated the role of T4 and T5 cells in flight behavior and found these cells involved in the detection of expansion motion. Flight avoidance turns as well as landing responses of flies depend on functional T4 and T5 cells. These behaviors are evoked by expansion motion like a looming stimulus, which mimics an approaching predator or object. The importance of T4 and T5 cells for looming evoked behavior suggests, that these cells are not only connected to lobula plate tangential cells, which respond to rotatory wide-field motion, but are also presynaptic to looming sensitive neurons in the lobula plate. The last manuscript describes transcription factors important for the differentiation of T4 and T5 neurons. The morphology of all T4 and T5 subtypes is comparable; their dendrites are oriented opposite to the preferred direction of the cell and the axon terminals target one of the four lobula plate layers. Both the dendrites and the axon terminals are limited to only one layer of their respective neuropil. We found two postmitotic transcription factors expressed in the young T4 and T5 cells, SoxN and Sox102F, which regulate the common features of all subtypes. These transcription factors are crucial for the proper morphology of the T4 and T5 cells, as well as the function of the adult neurons

Functional imaging of the neural components of Drosophila motion detection

In order to safely move through the environment, visually-guided animals use several types of visual cues for orientation. Optic flow provides faithful information about ego-motion and can thus be used to maintain a straight course. Additionally, local motion cues or landmarks indicate potentially interesting targets or signal danger, triggering approach or avoidance, respectively. The visual system must reliably and quickly evaluate these cues and integrate this information in order to orchestrate behavior. The underlying neuronal computations for this remain largely inaccessible in higher organisms, such as in humans, but can be studied experimentally in more simple model species. The fly Drosophila, for example, relies heavily on such visual cues during its impressive flight maneuvers. Additionally, it is genetically and physiologically accessible. Therefore, it is regarded as an ideal model organism for exploring neuronal computations underlying visual processing. During my PhD-thesis, I characterized neurons presynaptic to direction selective lobula plate tangential cells by exploiting the genetic toolbox of the fruit fly in combination with in-vivo imaging. The use of genetically encoded calcium indicators and two-photon microscopy allowed me to directly investigate response properties of small columnar neurons upstream of lobula plate wide field neurons. In the highly collaborative environment of our lab my imaging experiments were complemented by several other approaches, including electrophysiological and behavioral experiments, along with modeling which resulted in the publications that comprise this cumulative dissertation. Measuring calcium signals in T4 and T5 cells in the first study, established that both populations of neurons exhibit direction selective response properties. Furthermore, T4 cells only respond to moving bright edges, whereas T5 cells encode exclusively dark edge motion. Silencing the synaptic output of T4 and T5 separately, we were able to determine that both lobula plate tangential cell responses as well as the turning behavior of walking flies were impaired only to bright or dark edges, respectively. We thus proposed that the detection of the direction of visual motion must happen either presynaptic to, or on the dendrites of T4 and T5 neurons, and that this computation takes place independently for brightness increments and decrements. The second paper published in 2014 was motivated by an anatomical study that found an asymmetric wiring between L2 and L4 cells with the dendrites of Tm2 in the distal medulla. Using two-photon calcium imaging and neuronal silencing combined with postsynaptic electrophysiological recordings, we probed the contribution of L4 and Tm2 in the OFF pathway of Drosophila motion vision. We found that while Tm2 have small, isotropic, laterally inhibited receptive fields, L4 cells respond to both, small and large field darkening. Blocking the output of both cell types resulted in a strong impairment of OFF motion vision. In contrast to the anatomical prediction, we did not observe any directional effects for either of the cells

Review

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

Neural information processing in the Drosophila motion vision pathway

Detecting the direction of image motion is an essential component of visual computation. An individual photoreceptor, however, does not explicitly represent the direction in which the image is shifting. Comparing neighboring photoreceptor signals over time is used to extract directional motion information from the photoreceptor array in the circuit downstream. To implement direction selectivity, two opposing models have been proposed. In both models, one input line is asymmetrically delayed compared to the other, followed by a non-linear interaction between the two input lines. The Hassenstein-Reichardt (HR) model proposes an enhancement in the preferred direction (PD): the preferred side signal is delayed and then amplified by multiplying it with the other input signal. In contrast, the Barlow-Levick (BL) detector proposes a null direction (ND) suppression, whereby the null side signal is delayed and the other input is divided by it. The motion information is computed in parallel ON and OFF pathways. T4 and T5 are the first direction-selective neurons found in the ON and in the OFF pathway, respectively. Four subtypes of T4 and T5 cells exist each responding selectively to one of the four cardinal directions: front-to-back, back-to-front, upwards, and downwards, respectively. In the first manuscript, we found that both preferred direction enhancement and null direction suppression are implemented in the dendrites of all four subtypes of both T4 and T5 cells to compute the direction of motion. We, therefore, propose a hybrid model combining both PD enhancement on the preferred side and ND suppression on the null side. This combined strategy ensures a high degree of direction selectivity already at the first stage of calculating motion direction. Further processing, in addition to synaptic mechanisms on the dendrites of T4 cells, can improve the direction selectivity of the T4 cells' output signals. Such processing might involve: 1.) transformation from voltage to calcium, and 2.) from calcium to neurotransmitter release. In the second manuscript, we used 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 built a model which transforms T4 voltage responses into calcium responses. The model reproduced experimentally measured calcium responses across different visual stimuli using various temporal filtering steps and a stationary non-linearity. These findings provided a mechanistic underpinning of the voltage-to-calcium transformation and showed 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. The two manuscripts included in this thesis are presented chronologically and were published in peer-reviewed journals

The role of direction-selective visual interneurons T4 and T5 in Drosophila orientation behavior

In order to safely move through the environment, visually-guided animals use several types of visual cues for orientation. Optic flow provides faithful information about ego-motion and can thus be used to maintain a straight course. Additionally, local motion cues or landmarks indicate potentially interesting targets or signal danger, triggering approach or avoidance, respectively. The visual system must reliably and quickly evaluate these cues and integrate this information in order to orchestrate behavior. The underlying neuronal computations for this remain largely inaccessible in higher organisms, such as in humans, but can be studied experimentally in more simple model species. The fly Drosophila, for example, heavily relies on such visual cues during its impressive flight maneuvers. Additionally, it is genetically and physiologically accessible. Hence, it can be regarded as an ideal model organism for exploring neuronal computations during visual processing. In my PhD studies, I have designed and built several autonomous virtual reality setups to precisely measure visual behavior of walking flies. The setups run in open-loop and in closed-loop configuration. In an open-loop experiment, the visual stimulus is clearly defined and does not depend on the behavioral response. Hence, it allows mapping of how specific features of simple visual stimuli are translated into behavioral output, which can guide the creation of computational models of visual processing. In closedloop experiments, the behavioral response is fed back onto the visual stimulus, which permits characterization of the behavior under more realistic conditions and, thus, allows for testing of the predictive power of the computational models. In addition, Drosophila’s genetic toolbox provides various strategies for targeting and silencing specific neuron types, which helps identify which cells are needed for a specific behavior. We have focused on visual interneuron types T4 and T5 and assessed their role in visual orientation behavior. These neurons build up a retinotopic array and cover the whole visual field of the fly. They constitute major output elements from the medulla and have long been speculated to be involved in motion processing. This cumulative thesis consists of three published studies: In the first study, we silenced both T4 and T5 neurons together and found that such flies were completely blind to any kind of motion. In particular, these flies could not perform an optomotor response anymore, which means that they lost their normally innate following responses to motion of large-field moving patterns. This was an important finding as it ruled out the contribution of another system for motion vision-based behaviors. However, these flies were still able to fixate a black bar. We could show that this behavior is mediated by a T4/T5-independent flicker detection circuitry which exists in parallel to the motion system. In the second study, T4 and T5 neurons were characterized via twophoton imaging, revealing that these cells are directionally selective and have very similar temporal and orientation tuning properties to directionselective neurons in the lobula plate. T4 and T5 cells responded in a contrast polarity-specific manner: T4 neurons responded selectively to ON edge motion while T5 neurons responded only to OFF edge motion. When we blocked T4 neurons, behavioral responses to moving ON edges were more impaired than those to moving OFF edges and the opposite was true for the T5 block. Hence, these findings confirmed that the contrast polarityspecific visual motion pathways, which start at the level of L1 (ON) and L2 (OFF), are maintained within the medulla and that motion information is computed twice independently within each of these pathways. Finally, in the third study, we used the virtual reality setups to probe the performance of an artificial microcircuit. The system was equipped with a camera and spherical fisheye lens. Images were processed by an array of Reichardt detectors whose outputs were integrated in a similar way to what is found in the lobula plate of flies. We provided the system with several rotating natural environments and found that the fly-inspired artificial system could accurately predict the axes of rotation

Neuronal processing of translational optic flow in the visual system of the shore crab Carcinus maenas

This paper describes a search for neurones sensitive to optic flow in the visual system of the shore crab Carcinus maenas using a procedure developed from that of Krapp and Hengstenberg. This involved determining local motion sensitivity and its directional selectivity at many points within the neurone's receptive field and plotting the results on a map. Our results showed that local preferred directions of motion are independent of velocity, stimulus shape and type of motion (circular or linear). Global response maps thus clearly represent real properties of the neurones' receptive fields. Using this method, we have discovered two families of interneurones sensitive to translational optic flow. The first family has its terminal arborisations in the lobula of the optic lobe, the second family in the medulla. The response maps of the lobula neurones (which appear to be monostratified lobular giant neurones) show a clear focus of expansion centred on or just above the horizon, but at significantly different azimuth angles. Response maps such as these, consisting of patterns of movement vectors radiating from a pole, would be expected of neurones responding to self-motion in a particular direction. They would be stimulated when the crab moves towards the pole of the neurone's receptive field. The response maps of the medulla neurones show a focus of contraction, approximately centred on the horizon, but at significantly different azimuth angles. Such neurones would be stimulated when the crab walked away from the pole of the neurone's receptive field. We hypothesise that both the lobula and the medulla interneurones are representatives of arrays of cells, each of which would be optimally activated by self-motion in a different direction. The lobula neurones would be stimulated by the approaching scene and the medulla neurones by the receding scene. Neurones tuned to translational optic flow provide information on the three-dimensional layout of the environment and are thought to play a role in the judgment of heading