The Interactive Fly

Genes involved in tissue and organ development

  • The Adult Brain - Index to brain structure and function
  • Genes expressed in Adult Brain Development and Function
  • The Larval Brain and Development of the Adult Brain
  • Odor detection and Processing - Odorant receptors and olfactory receptor neurons, and olfactory learning
  • The Visual System - Optic lobe and optic glomeruli
  • The Central Body Complex - Ellipsoid body, superior arch, fan shaped body and the protocerebral bridge
  • Mushroom Bodies - The site of olfactory and other learning
  • Lateral Horn - Modulates other regions of the brain to ensure production of rapid and effective behavioral responses
  • Neuroendocrine complex - Ring gland: prothoracic gland, corpus allatum, and corpus cardiac
  • Subesophageal Ganglion - Site of the taste system and feeding behavior
  • Behavioral Paradigms - Sexual Behavior, motor Behavior, photoperiod response and others

    Optic glomeruli and their inputs in Drosophila share an organizational ground pattern with the antennal lobes

    Studying the insect visual system provides important data on the basic neural mechanisms underlying visual processing. As in vertebrates, the first step in visual processing in insects is through a series of retinotopic neurons. Recent studies on flies have found that these converge onto assemblies of columnar neurons in the lobula, the axons of which segregate to project to discrete optic glomeruli in the lateral protocerebrum. This arrangement is much like the fly's olfactory system, in which afferents target uniquely identifiable olfactory glomeruli. Whole-cell patch recordings show that even though visual primitives are unreliably encoded by single lobula output neurons because of high synaptic noise, they are reliably encoded by the ensemble of outputs. At a glomerulus, local interneurons reliably code visual primitives, as do projection neurons conveying information centrally from the glomerulus. These observations demonstrate that in Drosophila, as in other dipterans, optic glomeruli are involved in further reconstructing the fly's visual world. Optic glomeruli and antennal lobe glomeruli share the same ancestral anatomical and functional ground pattern, enabling reliable responses to be extracted from converging sensory inputs (Mu, 2012).

    Retinotopic output neurons from the lobula of Drosophila, which have axon diameters of 0.5 μm or less, do not transmit action potentials. This is typical of the many small interneurons in the insect visual system that are arranged as repeat ensembles. However, mushroom body intrinsic neurons (Kenyon cells) may be a special exception to this because very few of them, at any time, are required to accurately encode odorant identity through the mechanism of 'sparsening' (Mu, 2012).

    In the dipteran Phormia, relays connecting the medulla to the lobula and lobula plate have axon diameters of between 0.5 μm and 3 μm. These neurons generally respond with graded potentials as do the larger axon diameter (2–4 μm) lamina monopolar cells, which extend from the lamina to the medulla. Even the 15 μm diameter axons of 'giant' motion sensitive neurons in the lobula plate can conduct by graded potentials in addition to spiking responses. However, there are some exceptions. In the hoverfly Eristalis tenax, object-detecting neurons relaying from lobula show clear spiking responses, as do neurons in Phormia that respond to moving bars (Mu, 2012).

    Signal reliability is also critical for neurons that occur as single pairs of uniquely identifiable neurons, or as very small populations in the brain, or small subsets of Kenyon cells, each subset encoding an odorant. In Drosophila, such neurons conduct by spikes or by mixed codes: membrane potential fluctuations and action-potentials. Examples are the wide-field directional selective tangential cells of the lobula plate which occur either as a uniquely identifiable set of 3 HS neurons, or as 11 uniquely distinct VS neurons which collaborate to mediate responses to changes in optic flow. Neurons with long axons, such as the unique pairs of interneurons linking the central body with many areas of the lateral protocerebrum and deutocerebrum, or those which carry data from the brain to thoracic ganglia also invariably conduct by spikes (Mu, 2012).

    In contrast to uniquely identifiable pairs or small clones of neurons belonging to the midbrain, many neurons in the optic lobes occur as ensembles of identical, clonally related neurons. In the medulla of Drosophila and other fly species, there are about 50 types of retinotopic neurons, spaced one to each retinotopic colum. In the lobula, there are about 15 different clones of output neurons, each of which comprises an ensemble of about 40 identical neurons (Otsuna, 2006). Each neuron of an ensemble subtends 6–9 visual sampling units of the retina and has dendrites, and thus receptive fields (Okumura, 2007), that overlap with a surround of at least 8–12 neurons of the same clonal identity . This anatomical arrangement ensures that 8–12 neurons of the same clone view the same part of the visual field (Mu, 2012).

    In Drosophila, such outputs from the lobula have extremely thin axons and these cells conduct by graded potentials. Each LCN exhibits significant membrane-voltage fluctuations, which likely reflect the many postsynaptic sites from medulla afferents. An important finding of this study was that recordings from many single L1CNs show that none reliably encodes a visual primitive, whereas the summed responses of L1CNs show clear responses to defined visual stimuli. Thus, since any ensemble of LCNs converges at its unique glomerulus, it is expected that a subset of LCNs will respond to a given visual stimulus and that the summed responses of this subset would drive postsynaptic neurons of their target glomerulus. In larger dipterans, it has also been shown that different optic glomeruli respond to different visual primitives (Mu, 2012).

    It is conceivable that the weak responses recorded in individual LCNs is due to the long electrotonic distance between soma and axon. However there are two major reasons to reject the idea that these non-spiking characteristics are artifactual. Firstly, using an identical recording methodology, small spiking neurons in the mid brain were also shown to have long thin neurites between their cell body and their main integrative region. Secondly, recordings of the smallest retinotopic neurons in the medulla of a larger fly species, Phaenicia sericata, consistently showed that they encode data in a non-spiking fashion, irrespective of the location of the electrode in the neuron (Mu, 2012).

    If an individual output neuron from the lobula complex can have subtle and variable responses to specific visual stimuli, but the summed responses of a subset of LCNs belonging to the same clone show a clearer response, might local interneurons post-synaptic to their terminals in their relevant optic glomerulus integrate input signals and unambiguously respond to the same visual stimuli? Recordings of a LIN in the giant fiber optic glomerulus complex suggest this is case: the LIN responds unambiguously to a looming stimulus, whereas the response of the single type 2 lobula plate-lobula columnar neuron (LPL2CN) to the same stimulus can only be resolved from a power spectrum analysis. However, when responses of many of the same type of lobula output neurons are summed, their collective response is unambiguous (Mu, 2012).

    The giant fiber (GF) glomerulus receives LPL2CN inputs and contains LIN processes as well as one major dendritic process of the GF. The GF glomerulus glomerular local interneuron (LIN) responds to looming stimuli, and responds to intensity decrements. Looming stimuli activate the GF glomerulus's LPL2CN inputs. Responses by the LIN are also the same as those that drive the GF. That the LIN rapidly adapts to looming stimuli whereas GF does not suggests that several LINs are associated with the glomerulus and that these may recruit signals from successive groups of activated LPL2CN afferents. Though it remains to be demonstrated that the LPL2CN clone is presynaptic to the LIN and GF, there is strong evidence in larger dipteran species that the Col A afferents, which also converge on the GF glomerulus, are directly presynaptic to the GF. For example, electromicroscopy studies have shown that in Musca domestica, Col A cells establish electrical synapses onto the GF, and cobalt introduced into the GF passes, rather spectacularly, into the entire array of Col A afferents. Col A cells in the fly Phoenicia serricata respond with graded potentials to decrements in illumination and to movement of edges. The convergence of Col A neurons and neurons of the LPL2CN clone at the GF glomerulus does suggest that there is a more complex control system eliciting GF responses than has been hitherto envisaged (Mu, 2012).

    The convergence of axons from a clone of optic lobe outputs to an optic glomerulus suggests a mechanism that establishes reliable downstream responses: one or more local interneurons of the glomerulus complex integrate and average inputs from members of an isomorphic population of retinotopic relay neurons from the lobula complex. Recordings from the GF glomerulus show that its LIN responds reliably to the same looming stimulus that drives the LPL2CN afferent supply to that glomerulus. The demonstration that the LIN response is relatively noise-free, suggests that one function of LINs is to disambiguate information carried by afferents to a glomerulus, from synaptic noise generated at the dendritic trees within the lobula. Noise free information could then be relayed by the LIN to the glomerulus' projection neurons. These are of two types: premotor descending neurons, such as the GF, which project to the thoracic ganglion; and relay neurons which project to higher centers in the brain, such as the dorsal protocerebral lobes, and their connections to the central complex (Mu, 2012).

    This convergence of lobula outputs to uniquely identifiable optic glomeruli in the brain's first segment, the protocerebrum, is comparable to the convergence of olfactory sensory neurons (OSN) to antennal lobe glomeruli in the brain's second segment, the deutocerebrum, where each unique glomerulus in the fly's antennal lobe is targeted by the axons of a specific set of olfactory sensory neurons (OSNs) on the antenna, which expresses a particular olfactory receptor protein. In the antennal lobe, noisy signals from OSNs are refined by local interneurons and then relayed to higher centers by projection neurons. The present results provide electrophysiological evidences that noisy signals in an isomorphic population of lobula outputs are similarly refined by local interneurons of the optic glomerular complex. Therefore reliable responses in an optic glomerulus are established through convergence and signal averaging processes (Mu, 2012).

    The present studies further support the proposition that the optic glomerular complex and the antennal lobes are serially homologous neural systems having the same principle anatomical and functional organization, and with the common function of refining and integrating incoming signals (Strausfeld, 2007). Glomerular organization in the protocerebrum and deutocerebrum reflect a ground pattern that can be identified in every ganglion of the central nervous system. Throughout, each type of receptor, representing one or another modality, sends its axon to a specific domain in the relevant ganglion. These domains, in some ganglia represented by glomerular volumes, in others by allantoid or ovoid ones, are connected by spiking and non-spiking local interneurons which integrate the sensory input and relay behaviorally meaningful information to central neuropils and to motor circuits. Such arrangements evolutionarily derive from an ancestral ground pattern seen in archaic arthropods, each segment of which was composed of identical elements. As demonstrated by the protocerebrum and deutocerebrum, present day insects reflect this ancestral ground pattern even in the brain, despite each segment having evolved its unique sensory configuration (Mu, 2012).

    Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster

    In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. This study genetically dissected the achromatic motion channel in the Drosophila at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. This study showed that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior (Rister, 2007).

    Visual systems process the information from the environment in parallel neuronal subsystems. In higher vertebrates, for instance, the visual modalities of color, form, and motion are segregated at the level of the retina into separate channels. Similarly, insects have distinct sets of photoreceptors for motion and color. Investigating the motion channel in the Drosophila this study shows that at the next level below the eye, the lamina, the motion channel is again split into several functionally distinct parallel pathways (Rister, 2007).

    Directional responses to visual motion have been intensely studied, predominantly in dipteran flies. They are provided by arrays of elementary movement detectors, the smallest motion-sensitive units that temporally compare the intensity fluctuations in neighboring visual elements (sampling units; see Anatomy of Peripheral Interneurons of the Fly's Visual System). Their neuronal implementation in flies is still unknown. In the rabbit retina, a candidate interneuron computing directional motion has been identified (Euler, 2002). The present study is confined to the input side of the movement-detection circuitry (Rister, 2007).

    The compound eye of Drosophila is composed of about 750 ommatidia. Each of these contains eight photoreceptors (R1-8) that can be structurally and functionally grouped into two subsystems: six large photoreceptors (R1-6) mediate the detection of motion, whereas two small ones (R7, R8), together forming one rhabdomere in the center of the ommatidium, are required for color vision (Rister, 2007).

    The lamina consists of corresponding units called neuro-ommatidia, or cartridges. These are the sampling units of the motion channel, whereas the color channel (R7, R8) bypasses the lamina cartridge to terminate in the second neuropil, the medulla. The lamina is anatomically and ultrastructurally known in exquisite detail. Its functional significance, however, is little understood (Rister, 2007).

    In the lamina, the motion channel is split into four parallel pathways (see The Functional Role of L1 and L2 in Motion Detection). In each cartridge, the photoreceptor terminals are connected by tetradic synapses to four neurons, L1, L2, L3, and the amacrine cell α (amc; connecting to the medulla via the basket cell T1). The most prominent of these are the large monopolar cells L1 and L2. Their position in the center and their radially distributed dendrites throughout the depth of the cartridge suggest a key role in peripheral processing. This can be visualized by 3H-deoxyglucose activity labeling. Single-unit recordings of L1 and L2 in large flies so far have revealed only subtle differences between them. Their specific functional contribution to behavior is largely unknown (Rister, 2007).

    Several hypotheses have been advanced. The loss of L1 and L2 and concomitantly of optomotor responses in the mutant Vacuolar medullaKS74 had prompted a proposal that these cells were involved in motion detection. Later, however, it was claimed that L1 and L2 should be dispensable, because optomotor responses were still measured in flies that were assumed to have complete degeneration of L1 and L2 (Rister, 2007).

    If indeed L1 and L2 mediate motion vision, are they functionally specialized or redundant? The latter is unlikely to be the whole answer, considering the differing synaptic relationships of the two neurons. For one, they have their terminals in separate layers of the medulla. Second, L2, but not L1, has feedback synapses onto R1-6. These might play a role in neuronal adaptation and could exert a modulatory influence on the photoreceptor output. It has also been suggested that L1 and L2 might be specialized to provide the respective inputs to the two branches of the elementary motion detector (EMD) (Rister, 2007 and references therein).

    In Drosophila, L2 innervates and reciprocally receives input from a second-order interneuron, L4 that has two conspicuous backward oriented collaterals connecting its own cartridge to the neighboring ones along the x and y axes of the hexagonal array. In this network, the L2 neurons are connected to the L4 neurons of two adjacent cartridges, and the L4 neurons are directly connected to all six neighboring L4s. The significance of this circuitry is not yet understood. It has been speculated that the L4 network might be specialized for front-to-back motion, the prevalent direction in the visual flow-field of fast forward-moving animals (Rister, 2007).

    Using the two-component UAS/GAL4 system for targeted transgene expression, single interneurons or combinations of them, were manipulated in all lamina cartridges. To study whether a particular pathway was necessary for a given behavioral task, their synaptic output was blocked using the temperature-sensitive allele of shibire, shits1. In addition, the inverse strategy was adopted, studying whether single lamina pathways are sufficient for mediating the behavior in the same experimental context. Using a mutant of the histamine receptor gene outer rhabdomeres transientless (ort; Gengs, 2002) that has all lamina pathways impaired, the wild-type ort-cDNA was expressed in chosen types of lamina interneurons known to receive histaminergic input from R1-6. Testing necessity and sufficiency it is now possible to start to relate the structural organization of the lamina to visually guided behavior (Rister, 2007).

    This study reports the first steps into the genetic dissection of the neuronal circuitry mediating motion and position detection, the main perceptual processes of visual orientation behavior and gaze control. Some basic properties emerge: two subsystems, the L1 and L2 pathways, were identified that both mediate directional motion independently of each other. A third subsystem, the L3 pathway, may provide position information for orientation. The two motion pathways were remarkably redundant under a broad range of visual conditions, in line with the general observation that motion detection is a very robust phenomenon. To detect an impairment with only one of the pathways remaining intact, one had to drive the system to its operational limits (Rister, 2007).

    Clearly, the L1 and L2 pathways play the principal role in motion detection. Flies without the L3 and amc/T1 pathways are fully motion competent, as far as the present analysis can reveal. In contrast, flies with both L1 and L2 blocked are motion blind using optomotor yaw torque responses, motion-driven head movements, and landing response as criteria. This result is based on three independent driver lines and is in line with findings on the unmapped mutant VamKS74. As the L2 pathway mediates optomotor responses at very low stimulus strengths, it would not be surprising if few functional L2 neurons were sufficient to have mediated the response, like when there are few residual ommatidia in sine oculis mutant flies (Rister, 2007).

    The relation between the L1 and L2 pathways is of particular interest. Throughout most of the pattern contrast range either pathway alone provides full-sized motion responses. At high pattern contrast, the two pathways are redundant, while in the intermediate contrast range they are specialized for front-to-back and back-to-front motion, respectively. Only at the low end of the contrast range do the two pathways depend upon each other (Rister, 2007).

    In natural habitats of insects, intermediate pattern contrasts prevail. It is in this contrast range where the L1 and L2 pathways show unidirectional sensitivity for back-to-front and, respectively, front-to-back movement. A specialization of L1 and L2 for these two directions of motion had been proposed almost four decades ago. Different strengths of the respective optomotor responses in large flies and reduced responses for only one of the two directions in Drosophila mutants had suggested separate arrays of EMDs for the two directions. The new data are compatible with at least two models. In the first one, which is the sparser one, either neuron would serve its array of unidirectional EMDs: L1 an array for back-to-front, L2 one for front-to-back motion. The model would entail crosstalk between the two pathways at high pattern contrast, most likely in the medulla, and a more complex interaction between them at the low end of the pattern contrast range. The second model envisages EMDs for both directions to be served by either pathway. In this case, no crosstalk would be required at high pattern contrast, but one would be in need of additional explanations for the unidirectional responses in the intermediate contrast range (Rister, 2007).

    The asymmetry of the L4 collaterals and the close interaction between L4 and L2 are an intriguing structural correlate of the unidirectional contrast sensitivity of the L2 pathway. No equivalent network with opposite polarity has been detected in the lamina for the L1 pathway, but might still be found in the medulla. As long as no physiological data exist of L4 in Drosophila, it is not possible to tell whether the L4 network provides lateral inhibition, lateral pooling, or the second input pathway for an array of front-to-back EMDs (Rister, 2007).

    The L2 pathway is more sensitive to pattern contrast and low light intensity than the L1 pathway. As this distinction was observed with three independent genetic variants, an artifact due to the genetic methods is unlikely. The enhanced contrast sensitivity of L2 might be attributed to the feedback synapses of L2 onto photoreceptors R1-6, possibly providing some kind of gain control, or also to the L4 network. Enhancing sensitivity for front-to-back motion could be useful for fast flying animals, as this type of flow field prevails during fast forward flight. How these differences between the two pathways at low light intensity and pattern contrast show in flight behavior when both pathways are operating remains to be investigated (Rister, 2007).

    Somewhat surprisingly, the two lamina pathways seem not to be differentiated for speed or contrast frequency. Possibly, only one array of EMDs might exist for each direction (sparse model) and the two may have to be tuned the same. Genetic intervention in the lamina as studied here obviously does not affect the tuning of EMDs. This supports the view that motion processing is located proximal to the lamina (Rister, 2007).

    At high pattern contrast, the L1 and L2 pathways are redundant. L1 and L2 both mediate motion sensitivity in both directions. Bidirectionality at high contrast can be interpreted as crosstalk between two unidirectional pathways. This could be a property of the regular circuitry or due to wiring defects in the absence of neural activity in one of the pathways during development. The latter explanation is rather unlikely. In the L2-GAL4/shits1 flies, about equal back-to-front and front-to-back responses were observe at m = 10% pattern contrast, whereas the L1-GAL4 ort+ rescue flies at this pattern contrast respond only to back-to-front motion. Why should the permanently low neural activity in the L2 pathway during development (caused by the mutated histamine receptor) render an originally bidirectional L1 pathway more unidirectional (Rister, 2007)?

    The anatomical differences between the L1 and L2 pathways had prompted the speculation that the splitting of the signal from R1-6 into two pathways could correspond to the delayed and nondelayed input channels of the EMD. The present analysis refutes this idea as an overall explanation of the duplicity of the large lamina monopolar neurons. Either pathway alone mediates motion stimuli at high and intermediate pattern contrast. Hence, both neurons can serve the delayed as well as the nondelayed branch of the EMD. Yet, at the low end of the pattern contrast range of wild-type this is different. Neither L1 nor L2 alone mediate optomotor responses. The two pathways need to interact to provide motion sensitivity. Conceivably, by combining two unidirectional EMDs of opposite polarity one can more than additively improve their signal-to-noise ratio. Indeed, the original motion-detector model contains a subtraction of the signals of the two antidirectional EMDs to eliminate the dependency of the output upon light intensity. Alternatively, at this very low pattern contrast L1 and L2 might, after all, specialize to serve the delayed and respectively nondelayed branch of the EMD (Rister, 2007 and references therein).

    Finally, it is not yet clear whether the motion response based on the interaction of the L1 and L2 pathways operating at low pattern contrast is uni- or bidirectional. At the lowest contrast measuree (m = 5%), no directional preference was found in the control flies, although the overall response was already reduced to less than 50% (Rister, 2007).

    The high sensitivity for pattern contrast of the L2 pathway is paralleled by a low threshold for light intensity. At the lowest intensity measured at which wild-type is still responsive, the L2 pathway is not only necessary but also fully sufficient, implying again that under these conditions the L2 neurons serve both input channels to the EMD. It remains open whether at even lower intensities an interaction between L1 and L2 might be found as is the case with low pattern contrast (Rister, 2007).

    The data indicate that the special trade-off at low light intensity, whereby sensitivity is gained at the expense of acuity, can use the L1 pathway as input. The mechanism is supposed to pool the signals of many visual elements for the delayed as well as the nondelayed channels of an array of EMDs with large sampling base. In the current experiments, the L1 pathway at the broad pattern wavelength (λ = 36°) is about as sensitive as the L2 pathway at λ = 18°. This shows that the role of the L1 and L2 pathways in pooling is not yet understood well. Lower light intensities may reveal an involvement of also L2 in pooling (Rister, 2007).

    Recently, it has been shown that the T1 neuron has no conventional chemical synaptic output sites in the medulla as judged by its ultrastructure. Hence, it is an open question whether and how shits1 expression in T1 might block a presumed nonsynaptic output from T1. Expressing shits1 at the restrictive temperature has, on the other hand, been found to perturb the organization of microtubules in the expressing photoreceptor cells. Moreover, it is likely that the processing of other membrane vesicles and hormone secretion at the Golgi apparatus are affected as well. The data consistently show an effect of shits1 expression in T1 neurons at the restrictive temperature. Optomotor responses are reduced at intermediate pattern contrast, if L2 is blocked as well. The mechanism mediating this effect is not known (Rister, 2007).

    Assuming shits1 to block T1 output, it is concluded that the amc/T1 pathway supports the L1 pathway at intermediate pattern contrast, at which the response of the L1 pathway just reaches saturation. Under these conditions, disturbance of T1 reduces the gain of the system and shifts the saturation range to higher contrast levels. The finding that saturation is eventually reached could be explained by the assumption that neurons like L5 with a presumed higher response threshold might be added to the system at still higher pattern contrast. In line with this hypothesis is the finding that the on-off units in the outer chiasm of large flies, which might correspond to L5, did not respond to contrasts smaller than 10% in electrophysiological recordings. The rather subtle effect of blocking T1 is taken to indicate that the stimulus conditions for T1 function have not yet been properly defined. It is unlikely that T1 functions were not observed because shits1 did not block T1 output. Expression of DTI and Kir2.1 in T1 neurons did not show a more substantial effect (Rister, 2007).

    In contrast to earlier assumptions, evidence has been accumulating that orientation toward landmarks does not necessarily require motion. In Musca, position-sensitive torque responses could be elicited in stationary flight, if the luminance of a stationary vertical stripe was sinusoidally modulated (local flicker). In Drosophila, torque responses toward stationary dark objects (δ = 5°) have directly been documented (Rister, 2007 and references therein).

    In the present study, neuronal pathways mediating motion and position detection have been genetically separated. This study has shown that motion-blind animals are still able to approach landmarks, corroborating the notion that motion vision is not essential for the detection and fixation of a stationary object. In contrast, the data also suggest that motion detection improves the fixation of landmarks, especially when these are narrow or have a reduced contrast. Note, that in this paradigm testing freely walking flies motion vision was not excluded experimentally. Obviously, in visual orientation both neuronal subsystems are at work, and genetic dissection will help to unravel their interaction (Rister, 2007).

    In flies having the entire motion channel (R1-6) blocked, the color channel (R7/R8) alone provides basic position information. With only L1 and L2 blocked, flies are still completely motion blind in all paradigms tested, but their orientation behavior is distinctly superior to that of flies with the entire motion channel blocked. Apparently, elements among the remaining lamina pathways improve landmark orientation as mediated by R7 and R8. Given that L5 was blocked in one of the driver lines without an additional impairment of orientation behavior, it is assumed that at the conditions of the paradigm L5 did not substantially contribute to orientation behavior (Rister, 2007).

    Blocking T1 in addition to L1 and L2 caused no further reduction of the orientation response. Hence, the amc/T1 pathway seems not to contribute significantly to this behavior either. This means that the L3 pathway, possibly interacting with the R7 and R8 pathways in color vision, may mediate orientation behavior, since flies without functional L1, L2, and amc/T1 still show better orientation behavior than flies with the entire R1-6 channel blocked. The residual orientation behavior in flies without functional L1 and L2 is very sensitive to a reduction in object contrast. This suggests that the underlying phototactic or tropotactic orientation mechanism might integrate the visual input over large parts of the visual field, reducing the apparent pattern contrast of small targets below threshold. This spatial integration might occur at any level in the system (Rister, 2007).

    In summary, genetic dissection indicates that position detection might be as robust and redundant as motion vision. The color channel (R7/R8), L1, L2, and L3 all contribute to position detection. Presumably, single pathways are sufficient for this task. Detecting a singularity in space may require a less sophisticated neural mechanism than motion detection based on a temporal comparison of signals from neighboring visual elements (Rister, 2007).

    Applying circuit genetics, this study has found the peripheral neuronal network of the fly optic lobe is functionally more complex than what previous behavioral, anatomical, and electrophysiological studies on wild-type animals had revealed and, maybe, what the early pioneers of the 1950s and 1960s had envisaged. Still, with this new approach, the fly optic lobe once again proves to be a uniquely suited case study for gaining basic insights into the neuronal mechanisms of visual information processing and, more generally, for the comparison of structure and function in neural networks (Rister, 2007).

    ON and OFF pathways in Drosophila motion vision

    Motion vision is a major function of all visual systems, yet the underlying neural mechanisms and circuits are still elusive. In the lamina, the first optic neuropile of Drosophila melanogaster, photoreceptor signals split into five parallel pathways, L1-L5. This study examines how these pathways contribute to visual motion detection by combining genetic block and reconstitution of neural activity in different lamina cell types with whole-cell recordings from downstream motion-sensitive neurons. Reduced responses to moving gratings are found if L1 or L2 is blocked; however, reconstitution of photoreceptor input to only L1 or L2 results in wild-type responses. Thus, the first experiment indicates the necessity of both pathways, whereas the second indicates sufficiency of each single pathway. This contradiction can be explained by electrical coupling between L1 and L2, allowing for activation of both pathways even when only one of them receives photoreceptor input. A fundamental difference between the L1 pathway and the L2 pathway is uncovered when blocking L1 or L2 output while presenting moving edges of positive (ON) or negative (OFF) contrast polarity: blocking L1 eliminates the response to moving ON edges, whereas blocking L2 eliminates the response to moving OFF edges. Thus, similar to the segregation of photoreceptor signals in ON and OFF bipolar cell pathways in the vertebrate retina, photoreceptor signals segregate into ON-L1 and OFF-L2 channels in the lamina of Drosophila (Joesch, 2010).

    Neurons responding to visual motion in a directionally selective way are found in a vast number of animals and brain regions, ranging from the retina of rabbits to the visual cortex of macaques. In flies, large-field motion-sensitive neurons are located in the third neuropile layer, the lobula plate, and are thought to be involved in visual flight control. These lobula plate tangential cells are preferentially sensitive to vertical (VS cells) and horizontal (HS cells) motion, respectively. They depolarize when stimulated by motion along their preferred direction (PD motion) and hyperpolarize during motion along the opposite, null direction (ND motion). In the first neuropile, the lamina, photoreceptors R1-R6 provide input, directly or indirectly, onto five different monopolar cells (L1-L5) using histamine as their transmitter. L1-L5 send their axons into the medulla where neurons compute the direction of motion in accordance with the Reichardt model. Such motion detectors then provide excitatory and inhibitory input onto the dendrites of lobula plate tangential cells. However, the neural circuitry presynaptic to the tangential cells represented by the Reichardt detectors has so far escaped a detailed analysis, because of the small size of the columnar neurons. This study set out to elucidate the cellular implementation of the Reichardt model of visual motion detection starting from the lamina, asking which of the various neurons provide input to the motion detection circuitry. Previous studies addressing this question in Drosophila used behavioural read-outs to test for effects of blocking and rescuing of specific lamina cells. To get closer to the circuit in question, the Gal4 or Split-Gal4/UASsystem were used and genetic intervention was combined in different lamina neurons with electrophysiological recordings from lobula plate tangential cells (Joesch, 2010).

    Recordings were made from HS and VS cells, and the output of lamina neurons L1 and L2 was blocked by targeted expression of shibirets. Control flies always revealed strong and reliable responses to a moving grating, saturating for increasing contrast levels for both PD motion as well as ND motion. Blocking both L1 and L2 led to a complete loss of motion responses even at the highest pattern contrast. Blocking only L1 strongly reduced PD and ND responses for all contrasts tested. Blocking L2 using two different driver lines moderately reduced the responses at all contrast levels. To test whether the temperature shift alone could lead to altered motion responses, flies that had the same genotype as experimental flies except for the GAL4 driver gene were put to restrictive temperature 1 h before the experiment. The responses of these flies were indistinguishable from the ones of the other control flies. Together, these results indicate that L1 and L2 are necessary for wild-type responses to grating motion (Joesch, 2010).

    In a complementary approach, photoreceptor input to lamina cells L1 and L2 was selectively rescued via targeted expression of the wild-type histamine receptor, encoded by the ort gene, in an ort-null mutant background. Given the above results from the blocking experiments, rescuing either L1 or L2 pathway should lead to only small motion responses at best. However, rescuing L2 led to wild-type motion responses at all contrasts tested, for PD motion as well as for ND motion. The same was true when lamina cells L1 were rescued: again, motion responses were nearly indistinguishable from the ones of 'positive control' flies. In these positive control flies, no L1- or L2-GAL4, but one wild-type ort-allele, was present, leading to wild-type motion responses as expected. In 'negative control' flies, where either no L1-GAL4 and L2-GAL4 or no UAS-ort was present in an ort-null mutant background, motion responses were literally zero. Thus, blocking L1 or L2 revealed that the output of both L1 and L2 is necessary for wild-type motion responses. Rescuing the pathway of either L1 or L2 indicates, however, that either L1 or L2 is sufficient for a wild-type motion response. This contradiction deserves further investigation (Joesch, 2010).

    The blocking and rescuing experiments presented above differ in one important aspect: in one case, the synaptic output of L1 and L2 was blocked, in the other case, the synaptic input to the same cells was rescued. If L1 and L2 receive their input in parallel without any further interactions, both procedures should yield complementary results, which were not found. Thus, the existence of electrical connections between L1 and L2 was examined by immunolabelling of the innexin protein Shaking B, a member of the gap-junction-forming protein family in flies. Strong immunolabelling was found within the entire optic lobe including the lamina. Furthermore, the basal laminar processes of L1 and L2 appeared to co-localize with the Shaking B immunolabelling. Because some dipteran gap junctions were demonstrated to be permeable for neurobiotin, L1 cells were injected with neurobiotin and co-staining in L2 was looked for. When a single L1 cell was injected, a clear staining became visible in the adjacent L2 cell as well, identified by its characteristic terminal in medulla layer 2. Injecting L2 led to co-staining of the adjacent L1 cell, identified by its characteristic terminals in medulla layers 1 and 5. Therefore it is proposed that L1 and L2 are electrically coupled via gap junctions (Joesch, 2010).

    Gap-junctional coupling between L1 and L2 could, in principle, explain the contradictory results obtained in blocking and rescuing experiments: through electrical coupling, rescuing the photoreceptor input to L1 restores the L2 pathway as well, and vice versa. This explanation, however, requires that the coupling between L1 and L2 provides a sufficiently large input to the respective partner cell. To investigate the strength of the coupling, an inwardly rectifying potassium channel (Kir2.1) was expressed in one of the two lamina cells. When the potassium channel was expressed in L1 alone, motion responses were completely abolished, comparable to the situation when L1 and L2 were blocked by shibirets. A similar finding was obtained when the potassium channel was expressed in L2 cells. These results indicate a strong electrical coupling between L1 and L2 and, thus, resolve the apparent discrepancy between blocking and rescuing experiments (Joesch, 2010).

    So far, these data support the view that both L1 and L2 feed, with a somewhat different contribution, into the motion detection circuitry. However, no evidence is provided as to any functional specialization of each of the pathways. As one possibility, lamina cells L1 and L2 might be specifically involved in the analysis of either ON or OFF input signals, in analogy to the vertebrate retina. Because a grating stimulus is composed of many simultaneously moving dark-to-bright (ON edge) and bright-to-dark transitions (OFF edge), this would have escaped the analysis presented above. To investigate this possibility, moving edges of a single polarity were presented to flies in which the output of lamina cells L1 and L2 was blocked by shibirets. In control flies, moving ON and OFF edges elicited strong and reliable voltage responses in lobula plate tangential cells during PD and ND motion. When the output from L1 was blocked, the response to moving ON edges was literally zero whereas the response to moving OFF edges was still about 50% of the wild-type response. The opposite was true when the output from L2 was blocked by expressing shibirets using two different GAL4 driver lines: then, the response to moving ON edges was only mildly reduced whereas the response to moving OFF edges was nearly abolished (Joesch, 2010).

    In a pioneering study, and consistent with these results, it was found that rescuing either the L1 or the L2 pathway led to wild-type optomotor responses at high pattern contrasts. For low contrasts (5%-10%), a functional specialization of the L1 and L2 pathway for back-to-front and front-to-back motion was suggested, which, however, does not match the current data on tangential cell responses in that contrast range. The first evidence for a role of the L2 pathway in transmitting light OFF signals was obtained in a study on freely walking flies, where blocking L2 impaired turning tendencies in response to contrast decrements. However, the current finding that photoreceptor signals in the fly segregate into ON and OFF pathways via L1 and L2 neurons is surprising in so far as, different from ON and OFF bipolar cells of the vertebrate retina, both lamina cell types posses the same transmitter receptor and produce similar light responses in their dendrite. This similarity is likely to be increased even further by the gap-junctional coupling between dendritic compartments of L1 and L2, which might help to average out uncorrelated noise both cells receive from photoreceptor R1-R6 input. Subsequently, however, these signals must become differentially rectified. For L2, this has been recently shown to occur already within the cell, as L2 axon terminals reveal pronounced calcium signals selectively in response to light OFF stimuli (Joesch, 2010).

    Whether this also holds true for L1, or whether the selective responsiveness of the L1 pathway to light ON stimuli is only acquired further downstream in its postsynaptic neurons, is currently not known. On the basis of the co-stratification of columnar neurons as well as 2-deoxyglucose activity labelling, L1 and L2 have long been proposed to represent the entry points to two parallel motion pathways in the fly visual system, with L1 synapsing onto medulla intrinsic neuron Mi1 which in turn contacts T4 cells, L2 synapsing onto transmedullar neuron Tm1 which in turn contacts T5 cells, and with T4 and T5 cells finally converging on the dendrites of the lobula plate tangential cells (Joesch, 2010).

    The results provide evidence that these two pathways deal specifically with the processing of ON and OFF stimuli. Moreover, splitting a positively and negatively going signal into separate ON and OFF channels alleviates the neural implementation of a multiplication, as postulated by the Reichardt detector. Whereas otherwise, the output signal of the multiplier had to increase in a supra-linear way when both inputs increase as well as when they decrease, dealing with positive signals only in separate multipliers seems to be less demanding with respect to the underlying biophysical mechanism. Whatever this mechanism will turn out to be, the finding about the splitting of the photoreceptor signal into ON and OFF pathways adds to the already described commonalities between the invertebrate and the vertebrate visual system. Obviously, the selection pressure for an energy-efficient way of encoding light increments and decrements led to rather similar implementations across distant phyla (Joesch, 2010).

    Visualizing retinotopic half-wave rectified input to the motion detection circuitry of Drosophila

    In the visual system of Drosophila, photoreceptors R1-R6 relay achromatic brightness information to five parallel pathways. Two of them, the lamina monopolar cells L1 and L2, represent the major input lines to the motion detection circuitry. A new method was devised for optical recording of visually evoked changes in intracellular Ca2+ in neurons using targeted expression of a genetically encoded Ca2+ indicator. Ca2+ in single terminals of L2 neurons in the medulla carried no information about the direction of motion. However, this study found that brightness decrements (light-OFF) induced a strong increase in intracellular Ca2+ but brightness increments (light-ON) induced only small changes, suggesting that half-wave rectification of the input signal occurs. Thus, L2 predominantly transmits brightness decrements to downstream circuits that then compute the direction of image motion (Reiff, 2010).

    The fly visual system continuously provides information about the motion of objects, conspecifics, predators and the three-dimensional structure of the environment. This information underlies the execution of notable visually driven behaviors. However, the manner in which small-scale neural networks accomplish such computational efficacy remains an open question, and the complete motion detection circuitry has not yet been determined in any animal. This study examined this question in Drosophila by analyzing how brightness changes become encoded in changes in the concentration of presynaptic Ca2+ in the axon terminals of L2 neurons, a major input channel to the motion detection circuitry (Reiff, 2010).

    The processing of brightness changes underlies the detection of visual motion. On the basis of a detailed input-output analysis of the optomotor response in tethered beetles, the well-known Hassenstein-Reichardt model (HRM) of visual motion detection was derived. The HRM essentially performs a spatio-temporal cross-correlation of two luminance input signals by multiplying the signals derived from two neighboring image points after one of them has been temporally delayed. This operation is executed in each of two mirror-symmetrical half-detectors that operate with opposite sign. Summing the output of both half-detectors results in a directionally selective response of the full detector. Notably, the HRM precisely describes the observed optomotor behavior of walking beetles and walking and flying flies in algorithmic terms. Furthermore, the fundamental computations of the HRM can explain motion detection in different vertebrate species, including humans. In flies, directionally selective responses that closely match the predictions of the model are observed in the large tangential neurons of both large fly species and Drosophila. These cellular responses carry distinct signatures that derive from the correlative processing in the HRM12 (Reiff, 2010).

    Because of the purely algorithmic nature of the HRM, no immediate conclusions about the underlying neuronal hardware can be drawn; different implementations of the model can result in similar output. To gain insight into the cellular implementation of the model, extracellular responses of the directionally selective H1 neuron where recorded while presenting apparent motion stimuli. Results of sequential stimulation of individual photoreceptor pairs (R1 and R6) of the same ommatidium led to the proposal that each input signal is split into an ON and an OFF channel that then fed into separate multipliers for the processing of brightness increments and decrements, respectively. However, interactions among brightness increments and decrements are inherent in the original HRM and have repeatedly been observed in behavioral optomotor responses and in the cellular responses of the H1 neuron21. Apparent motion stimuli with opposite polarity induce responses that report a reversal of the true direction of the stimulus, a phenomenon that is known in psychophysics as reverse-phi. If a neuron is assumed to perform a multiplication in a sign-correct manner, then this neuron's output signal should increase in a supra-linear way when both inputs increase as well as when both inputs decrease. No biologically plausible mechanism is known that could accomplish such a computation. A circuit was proposed that was inspired by the 'four-quadrant multiplier' used in analog signal processing: bipolar (both positive and negative signal components) input signals were half-wave rectified, resulting in only positive signals. These signals are subsequently processed in four separate multipliers accounting for all possible interactions (ON-ON, ON-OFF, OFF-ON and OFF-OFF). The output of the individual multipliers is then summed by a postsynaptic integrator in a sign-correct manner (from the perspective of this integrator). Thus, in contrast with a previous account, separate input channels for the processing of brightness increments and decrements cannot be excluded on the basis of responses of the integrating neuron to mixed input signals (Reiff, 2010).

    In flies, the lamina monopolar neurons L1 and L2 are the largest and best-investigated second-order visual interneurons postsynaptic to the photoreceptors R1-R6. L1-L3 and one amacrine cell (amc) all express a chloride channel encoded by the ort gene, which is gated by the photoreceptor transmitter histamine. The processes of amacrine cells stay in the lamina, where they synapse onto L5 and where L4 receives input from L2 (and feeds back onto two more lateral L2 neurons). L4 and L5, as well as L1-L3, project to distinct layers in the medulla. Thus, five possible parallel processing streams (three direct channels, L1-L3; two indirect channels, L4 and L5) transmit information about brightness changes from the lamina to the medulla. Behavioral and genetic experiments suggest that L3 is involved in processing of ultraviolet light and in phototaxis. In contrast, L1 and L2 provide the major input to the motion detection circuitry in the medulla. Recordings of their dendritic membrane potential reveal nondirectional, strongly adapting responses in large fly species; dendritic voltage changes in L1 and L2 to a transient light pulse correspond to an inverted, high pass-filtered biphasic version of the voltage change recorded in photoreceptors that depolarize in response to light. The reported inhibitory current through histamine-gated chloride channels explains the hyperpolarizing ON response; however, it does not explain the excitatory OFF component at the end of a light pulse that has been observed in large flies. Depolarizing voltage responses to light-OFF have not been observed in Drosophila (Reiff, 2010).

    Even though there have been electrophysiological studies on lamina monopolar cells and few other columnar neurons in Calliphora, the signals that are transmitted by lamina monopolar cells to neurons of the motion detection circuitry in the medulla could not be recorded so far for methodological reasons. This study addresses this problem in fly motion vision by investigating how L2 axon terminals in the medulla encode brightness changes in presynaptic intracellular calcium. Visually evoked Ca2+ is measured by a new method that employs optical recording of the genetically encoded calcium indicator TN-XXL targeted to L2 neurons and an interlaced visual stimulation technique (Reiff, 2010).

    The data on L2 terminal Ca2+ corroborate the previously reported inversion and high pass-filtering and complete the processing by adding half-wave rectification of the brightness signal. Taking into account the role of Ca2+ in presynaptic vesicle release, it is proposed that L2 primarily transmits the information about brightness decrements to the motion detection circuit in the medulla (Reiff, 2010).

    Dendritic recordings of L2 membrane potential in large flies show at least small depolarizing responses induced by light-OFF, suggesting that the underlying processing likely involves an amplification of the positive dendritic membrane potential and opening of voltage-activated Ca2+ channels in L2 terminals induced by light-OFF. Light-ON hyperpolarizes the L2 membrane potential, which might rapidly inactivate the calcium channels. Efficient calcium extrusion then likely mediates the observed rapid return of the calcium signal to baseline that is induced by light-ON (Reiff, 2010).

    Nonlinear processing steps represent an important feature of second-order visual interneurons in flies and in the vertebrate retina; vertebrate ON- and OFF- bipolar cells preferentially relay either increments or decrements in brightness. However, half-wave rectification in bipolar cells is not complete and partly results from inhibitory interactions among ON and OFF channels. The increase of the time constant observed in L2 rescue flies suggests that interactions between different lamina cell types are involved in the generation of imperfectly half wave-rectified light-OFF calcium responses in L2 axon terminals. Such interactions are also suggested by the rich anatomical connections at the level of the dendrites in the lamina and at the level of the axon terminals in the medulla. Furthermore, given that L2 terminals transmit their main signal at light-OFF, other channels must exist for the signaling of brightness increments. Such ON and OFF signaling is a common motif in different animals and sensory modalities. Thus, although not necessary for Hassenstein-Reichardt-type computations, half-wave rectifying the input signals into parallel ON and OFF channels and multiplying each pair separately allows the outputs to be treated in a sign-correct manner. The devised imaging approach should pave the way for future studies that ultimately reveal the cellular implementation of the HRM of visual motion detection (Reiff, 2010).

    The color-vision circuit in the medulla of Drosophila

    Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green-sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Ommatidia exhibit unique fluorescence of their inner photoreceptors that appear either yellow (y) (70% of ommatidia) or pale (p) (the remaining 30%). The p-ommatidia contain UV-Rh3 in R7 and blue-Rh5 in R8, whereas y-ommatidia contain UV-sensitive Rh4 in R7 and green-sensitive Rh6 in R8. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. The neural network in the medulla was reconstructed, focusing on neurons likely to be involved in processing color vision. The full complement of neurons in the medulla was identified, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. Third-order neurons and local neurons were identified that likely modulate information from second-order neurons. Finally, highly specific tools are presented that will allow functional manipulation the network and test both activity and behavior. This precise characterization of the medulla circuitry will facilitate understanding of how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates (Morante, 2008).

    The medulla represents the major neuropil in the optic lobe. Despite this complexity, by using a series of TFs-Gal4 lines, the medulla network has been dissected at the single-cell level. ap- and ey-Gal4 are expressed in nonoverlapping populations, both in projection and local neurons. dll-Gal4 reveals expression almost exclusively in local neurons, although not all local neurons are marked by dll-Gal4. Through this extensive analysis it has been possible to reconstruct morphologically 38 types of projection neurons, 22 types of local neurons and 3 connecting neurons. Among these cell types, six new projection and four local neuron types not described before have been identified. Most of these new cell types (e.g., TmLM7 and PmLM7) include cells with ramifications exclusively in the lower medulla domain (Morante, 2008).

    It is thus possible to define the elements represented in a 'column,' the medulla functional units: (1) two inputs from R7 and R8, (2) five ramifications from L1-L5 lamina neurons, (3) 11 types of columnar and 20 of noncolumnar projection neurons, (4) four types of columnar and 11 of noncolumnar local neurons, and (5) two types of columnar and one of noncolumnar lamina connecting neurons. Additionally, seven other noncolumnar local and seven projections neurons do not contact PRs. Overall, each R7/R8 termination pair is therefore surrounded by at least 54 different cell types, with 14 other cell types that do not contact PRs. The enormous number of cell types forming part of a column contrasts with the relatively small number of elements that feed into the medulla (R7-R8 and R1-R6 through L1-L5 neurons). Thus, the divergence in the flow of information between PRs and medulla neurons argues that much local processing occurs in the medulla. In contrast, in the deeper optic lobe (i.e., lobula and lobula plate), the number of cells and their wide-field ramifications argue for the convergence of information from medulla neurons (Morante, 2008).

    This analysis of the color-vision network in the Drosophila medulla reveals the presence of two parallel routes carrying and processing visual information that coexist in the medulla neuropil: (1) a point-to-point pathway formed by columnar neurons that receive information from only a single ommatidium ('vertical integration'), and (2) a pathway with a broader receptive field composed by noncolumnar neurons that receive information from photoreceptors from several ommatidia ('horizontal integration') (Morante, 2008).

    The first pathway integrates outputs from R8 and R7, likely allowing broad wavelength discrimination between UV- and blue or green: cells comparing signals from p-ommatidia might mediate better discrimination among short wavelengths, whereas those comparing outputs of y-photoreceptors should better mediate discrimination among longer wavelengths. This suggests that there are two independent and nonoverlapping retinotopic maps that separately process color information: one from p- and one from y-photoreceptors. These maps might be physically separated in higher brain centers, but specific p- or y-contacting neurons are not observed, are distinct projections to different layers in the lobula complex seen (Morante, 2008).

    The second pathway reflects more complex visual processing, with noncolumnar Tm neurons cells integrating information from several R8 and R7 photoreceptors. Because the p- and y-ommatidia each compare output of photoreceptors with widely different absorption spectra (UV and green, or UV and blue), 'horizontal integration' between y- and p-ommatidia for both R7 and R8 might allow a much more precise evaluation of the colored world, although at a reduced spatial resolution. Additionally, cells were found that might directly compare p- and y-R8 cells (e.g., contacting blue and green R8, but not R7). Similarly, cells that compare p- and y-R7 photoreceptors might integrate information from the two different types of UV-photoreceptors. It should be noted that the difference in peak of absorption between Rh3 and Rh4 (~20 nm) in R7 is sufficient to allow precise discrimination of UV wavelengths and is similar to the difference between M- and L- opsins in humans (30 nm). Thus, this horizontal integration should allow the convergence of several inputs from multiple R7 and R8 photoreceptors to a single medulla cell (Morante, 2008).

    It is not clear how comparison between photoreceptors is achieved and whether local neurons expressing different neurotransmitters are involved to generate opposite outputs between R7 and R8 (for columnar neurons) or between p- and y-R7 or R8 (for noncolumnar neurons). It is likely that R7 cells do sum up their output to support the strong UV attraction that characterizes flies. However, inner photoreceptors are not involved in scotopic vision (dim light), and an organization in which neurons simply add their outputs makes little sense for what is known of the function and specialization of R7 and R8 cells in color vision. Therefore, it is likely that an opponent system exists in the medulla and that it is mediated by local neurons. Alternatively, color vision might not need an opponent system but might result from nonlinear interactions between R7 and R8. Their interaction with the same postsynaptic cell could be complex, e.g., Tm neurons might have synergistic responses to inputs from R7 and R8 or from photoreceptors from different ommatidia (Morante, 2008).

    In the mammalian retina, horizontal and amacrine cells are interneurons that have a critical role in modulating retinal output. Horizontal cells provide lateral interactions between photoreceptor terminals, creating a 'center-surround organization,' enhancing the response of ganglion cells lying directly under the light stimulus and inhibiting their neighbors. Meanwhile, amacrine cells not only make inhibitory synapses on bipolar cells, thus controlling their output to ganglion cells, but also synapse onto ganglion cells and coordinate their firing. As in the mammalian retina, a great variety of neurons with local ramifications within the medulla might modulate the visual outputs carried by projection neurons. This modulation is accomplished by two kinds of local neurons. (1) Columnar local neurons with one Mi cell per ommatidium. Interestingly, the arborizations of many of these columnar local neurons resemble those of corresponding Tm columnar projection neurons that contact either one photoreceptor, or both R7 and R8. They intermingle with them in photoreceptor layers as well as in lower medulla layers. In this microcircuit, Mi cells appear to interact both pre- and post-synaptically with Tm ramifications. (2) Dm cells are noncolumnar local neurons that do contact photoreceptor layers, whereas Pm cells only have projections in lower medulla layers. Both classes ramify extensively over several columns. Interestingly, noncolumnar local neurons appear to be pre- and post-synaptic both in photoreceptors layers and in lower medulla layers. This suggests that Dm local neurons perform a first level of integration with Tm cells at the level of photoreceptor layers, whereas this information is further processed by Pm cells that act at a second level in lower medulla layers (Morante, 2008).

    Therefore, columnar and noncolumnar projection neuron outputs could be modulated by columnar and noncolumnar local neurons, respectively. In the Drosophila antennal lobe, two kinds of local neurons exist: inhibitory local interneurons and a local excitatory population involved in processing projection neurons signals to downstream targets. A similar system might also exist in the medulla (Morante, 2008).

    Whether Tm projection neurons are columnar or noncolumnar, they all arborize in lower medulla layers (M7-M10). For most Tm cells analyzed, these lower medulla ramifications appear to contain both presynaptic and postsynaptic terminations, suggesting that this region represents a second layer of integration in the color-vision pathway after the direct comparison between R7 and R8 (or between y and p R7/R8) (Morante, 2008).

    A class of projection neurons, TmLM7 and TmLM8 only arborize in lower medulla layers, where they mostly exhibit postsynaptic arborizations. This suggests that they play a role as third-order neurons that collect more elaborate visual information already integrated by other Tm cells with ramifications in photoreceptor layers and presynaptic endings in lower medulla layers and then carry this processed visual information to downstream targets (Morante, 2008).

    These observations have allowed reconstruction of the organization of the visual circuit in Drosophila. Generating single-cell clones allowed deciphering of many of the intricacies of this pathway and have led to the proposal of general rules of color-vision processing in the medulla and transmission to downstream targets in the deeper optic lobes. Additionally, Gal4 lines have been idneitifed with very restricted expression patterns in neuronal subtypes in the medulla. Future electrophysiological and behavioral experiments using these and additional Gal4 lines will help reveal the exact function of these optic lobe cells in these complex circuits and to reach a better understanding of the mechanisms that govern the physiology of vision both in invertebrates and vertebrates (Morante, 2008).

    The neural substrate of spectral preference in Drosophila

    Drosophila vision is mediated by inputs from three types of photoreceptor neurons; R1-R6 mediate achromatic motion detection, while R7 and R8 constitute two chromatic channels. Neural circuits for processing chromatic information are not known. This study identified the first-order interneurons downstream of the chromatic channels. Serial EM revealed that small-field projection neurons Tm5 and Tm9 receive direct synaptic input from R7 and R8, respectively, and indirect input from R1-R6, qualifying them to function as color-opponent neurons. Wide-field Dm8 amacrine neurons receive input from 13-16 UV-sensing R7s and provide output to projection neurons. Using a combinatorial expression system to manipulate activity in different neuron subtypes, it was determined that Dm8 neurons are necessary and sufficient for flies to exhibit phototaxis toward ultraviolet instead of green light. It is proposed that Dm8 sacrifices spatial resolution for sensitivity by relaying signals from multiple R7s to projection neurons, which then provide output to higher visual centers (Gao, 2009).

    Many animals respond differentially to light of different wavelengths: for example, most flying insects exhibit positive phototactic responses but prefer ultraviolet (UV) to visible light, whereas zebra fish are strongly phototactic to ultraviolet/blue and red light but weakly to green. Unlike true color vision, which distinguishes lights of different spectral compositions (hues) independently of their intensities, spectral preferences are strongly intensity-dependent and innate, probably reflecting each species' ecophysiological needs. Thus, water fleas (Daphnia magna) avoid harmful UV but are attracted to green light, which characterizes abundant food sources. Daylight is rich in UV, so flying insects' preference for UV over visible light is probably related to the so-called open-space response, the attraction towards open, bright gaps and away from dim, closed sites. The receptor mechanisms for spectral preference has been well studied in flying insects, especially in Drosophila. Two or more photoreceptor types with distinct spectral responses are required to detect different wavelengths of light, and mutant flies lacking UV-sensing photoreceptors exhibit aberrant preference for green light. However, the post-receptoral mechanisms of spectral preference are entirely unknown. Furthermore, it is not clear how spectral preference is related to true color vision. Color-mixing experiments suggest that color vision spectral preference are independent in honeybees. In Drosophila, however, spectral preference experiments have revealed that the phototactic response towards UV is significantly enhanced by the presence of visible light, suggesting a 'color' contrast effect in spectral preference behavior. Identifying and characterizing the neural circuits that process chromatic information is the first step to understanding the post-receptoral mechanisms of spectral preference and thus color vision (Gao, 2009).

    With recent advances in genetic techniques that manipulate neuronal function, Drosophila has re-emerged as a model system for studying neural circuits and functions. In particular, the Gal4/UAS expression system combined with the temperature-sensitive allele of shibire makes it possible to examine the behavioral consequences of reversibly inactivating specific subsets of neurons. Such interventions allow direct comparisons between the connections of a neuron and its function, thereby establishing causality (Gao, 2009).

    The Drosophila visual system comprises the compound eye and four successive optic neuropils (lamina, medulla, lobula and lobula plate. The compound eye itself has some 750 ommatidia, populated by two types of photoreceptors. The outer photoreceptors R1-R6, which are in many ways equivalent to vertebrate rod cells, express Rh1 opsin and respond to a broad spectrum of light, and are thus presumed to be achromatic. The inner photoreceptor neurons R7 and R8 have complex opsin expression patterns: R7s express one of two ultraviolet (UV)-sensitive opsins, Rh3 and Rh4, while beneath R7 the R8s coordinately express blue-sensitive Rh5 or green-sensitive Rh6 opsins. The achromatic R1-R6 channel mediates motion detection. R1-R6 innervate the lamina, where the achromatic channel input diverges to three or more pathways mediated by three types of lamina neurons, L1-L3. Their synaptic connections have been analyzed exhaustively at the electron microscopic (EM) level. Genetic dissection indicates that these three pathways serve different functions in motion detection and orientation. Much like vertebrate cones, R7 and R8 photoreceptors are thought to constitute chromatic channels that are functionally required for spectral preference behaviors. The axons of R7 and R8 penetrate the lamina and directly innervate the distal medulla, where until now their synaptic connections have been completely unknown (Gao, 2009).

    The medulla, the largest and most heavily populated optic neuropil, is organized into strata (M1-M10) and columns, in a manner reminiscent of the mammalian cortex. All visual information converges upon the distal strata of the medulla: the axons of R7 and R8 directly innervate strata M6 and M3, respectively, while L1-L3 transmit information from the R1-R6 channel to multiple medulla strata (M1/5, M2, and M3, respectively). The R7, R8, and L1-L3, which view a single point in visual space innervate a single medulla column and there establish a retinotopic pixel. Previous Golgi studies have revealed about 60 morphologically distinct types of medulla neurons. Each arborizes in a stereotypic pattern within specific strata of the medulla, and projects an axon to a distinct stratum of the medulla, lobula or lobula plate. The distinct morphological forms of different types of medulla neurons reflect, at least in part, their diverse patterns of gene expression. Although it is widely presumed that the medulla incorporates key neural substrates for processing color and motion information, little is known about its synaptic circuits and their functions. EM analyses of synaptic circuits have not been possible because of the complexity of this neuropil, while electrophysiological investigations are technically challenging because of the small size of neurons (Gao, 2009).

    This study investigated the chromatic visual circuits in the medulla. Using a combination of transgenic and histological approaches, the first-order interneurons in the medulla that receive direct synaptic inputs from the chromatic channels, R7 and R8 were identified. These neurons were subdivided based on their use of neurotransmitters and gene expression patterns. By systematically inactivating and restoring the activity of specific neuron subtypes, the neurons that are necessary and sufficient to drive a fly's phototactic preference to UV were identified (Gao, 2009).

    Previous electrophysiological and histological studies have demonstrated that Drosophila photoreceptor neurons are histaminergic and that R7 and R8 photoreceptors provide the predominant histamine-immunoreactive input to the medulla. Two ionotropic histamine-gated channels, Ort (ora transientless; HisCl2) and HisCl1 have been identified. Mutants for ort exhibit defects in motion detection and their electroretinograms (ERGs), indicating that Ort is required to transmit R1-R6 input to the first-order interneurons (Gengs, 2002). To test whether Ort is required for visually guided behavior, flies' phototaxis towards either UV or green light in preference to dark was examined. This phototactic response is mediated primarily by the more sensitive, broad-spectrum photoreceptors, R1-R6, although R7 cells also contribute to UV, but not green, phototaxis under the light-adapted condition. Wild-type flies exhibit stronger phototaxis towards UV than towards green light by approximately one order of magnitude, and light-adaptation, when compared with dark-adaptation, reduces the sensitivity to UV and green light by approximately two orders of magnitude. In contrast, strong transallelic combination ort1/ortUS2515 mutant flies exhibit much weaker phototaxis towards either UV or green light (by about three and two orders of magnitude, respectively) as compared with wild-type. In negative geotaxis assays, ort mutants exhibit no apparent motor defects, suggesting that their reduced phototaxis was not a motor system defect but rather a visual deficit. In addition, the ort mutation affects UV phototaxis more severely than green phototaxis. It is speculated that Ort plays a role in relaying signals from UV-sensing R7s to their first-order interneurons, and that HisCl1 may participate in phototaxis, especially towards green light (Gao, 2009).

    To assess whether Ort is required to transmit chromatic input mediated by R7 and R8, a quantitative spectral-preference assay was used. This spectral-preference assay tests the phototaxis towards UV in preference to green and depends on R7, but not significantly on R1-R6, function. This behavior depends on the circuit comparing UV and green light and likely reflects salience of UV and green lights rather than a simple linear summation of their phototactic responses. It was found that wild-type flies prefer short-wavelength UV to longer-wavelength green light in an intensity-dependent fashion. In contrast, homozygous null ort1 mutants and strong transallelic combination ort1/ortUS2515 mutants (as well as other allelic combinations, ortP306/ortUS2515 and ort1/ortP306) all exhibited reduced UV preference. Over five orders of magnitude in the ratio of UV/green intensities, the proportion of ort mutant flies that chose UV was significantly lower than that for wild-type flies. To quantify the UV preference, the isoluminance point, the UV/green intensity ratio at which flies found light of either wavelength equally 'attractive', was determined, and the negative logarithm of the intensity ratio was used as a measure of UV attractiveness. The UV attractiveness for ort mutants was significantly lower than that for wild-type flies (AttrUV/G=2.52±0.23) but higher than that for sevenless mutants (Gao, 2009).

    Given that ort null mutants still exhibits phototaxis, whether the other histamine receptor, HisCl1, might contribute to UV preference, was examined. HisCl1134 null mutants were found to exhibit UV preference indistinguishable from the wild-type. In contrast, strong allelic combination HisCl1134 ort1/HisCl1134 ortP306 double-mutants shows weak phototaxis towards green light, while double-null HisCl1134 ort1 mutants, like the phototransduction mutant NorpA, fails to discriminate between wavelengths in the UV and green. It is concluded that Ort is essential for optimal UV preference while HisCl1 plays at most a minor and partially redundant role. It is noted that double-null HisCl1134 ort1 mutants are not entirely blind and still exhibit very weak fast phototaxis, suggesting that there might be residual synaptic transmission between photoreceptors and the first-order interneurons despite of the absence of these two known histamine receptors (Gao, 2009).

    It was reasoned that the first-order interneurons must express the histamine receptor Ort in order to respond to their inputs from histaminergic R7 and R8 terminals (see The histamine chloride channel Ort is expressed in subsets of lamina and medulla neurons). To identify these first-order interneurons, the ort promoter region was determined using comparative genomic sequence analysis (Odenwald, 2005; Yavatkar, 2008). In the ort locus, four blocks of non-coding sequence were found that are highly conserved among 12 species of Drosophila. The first three sequence blocks (designated C1-C3) are localized to the intergenic region and the first intron and are therefore likely to contain critical cis-elements. ort-promoter constructs driving Gal4 or LexA::VP16 were generated, designating these ortC1-3-Gal4 and ortC1-3-LexA::VP16. Both driver systems drove expression patterns in identical subsets of neurons in the lamina, medulla cortices and in the deep C and T neurons of the lobula complex, except that ortC1-3-Gal4 drove somewhat patchy expression with lower intensity. The fourth block of conserved sequences, located at 3'UTR, contains putative microRNA binding sites and, as examined in ortC1-4-Gal4, did not drive expression in additional cells, suggesting that it does not contain critical cis-elements. Overall, the expression patterns of these ort promoter constructs resembled previously published ort expression patterns (Witte, 2002) from in situ hybridization (Gao, 2009).

    Comparative genomic sequence analysis was also performed for the HisCl1 locus and two blocks of highly conserved sequence (C1 and C2), located in the first introns of the HisCl1 gene and its neighboring gene (CG17360) were identified. A HisCl1-Gal4 construct was generated that included these conserved sequences. It was found that HisCl1-Gal4 drove strong expression in the lamina epithelial glia cells (as recently also reported by Pantazis (2008) and medulla cells that are not well characterized. This result is consistent with previous EM data that lamina epithelial glia enwrap each cartridge and are postsynaptic to R1-R6. Insofar as both the behavioral requirement and expression pattern indicate that Ort but not HisCl1 plays a critical role in the visual system, focus was placed on Ort in the following analyses (Gao, 2009).

    Whether using the ort-promoter Gal4 drivers to express Ort is sufficient to rescue defects in the visual behavior ort mutants was examined. It was found that ortC1-4-Gal4-driven Ort expression restored a preference for UV (AttrUV/G=2.25±0.34) in ort mutants to the wild-type level. Since Ort, but not HisCl1, is also required in lamina neurons for normal ERG and motion detection responses (Gengs, 2002; Rister, 2007), the rescued flies were examined for these functions too. It was found that expressing Ort in ort mutants using ortC1-3-Gal4 restored, at least qualitatively, the 'on'- and 'off'-transients of the ERG, which report transmission in the lamina, as well as the optomotor behavior. These findings are consistent with the observation that ort-Gal4 drives reporter expression in lamina neurons L1-L3. In contrast, expressing Ort in lamina neurons L1 and L2 using an L1/L2-specific driver (L1L2-A-Gal4) rescued both the ERG, at least qualitatively, and optomotor defects, but not the UV preference of ort mutants. Thus, the actions of the ort-Gal4 drivers recapitulated the endogenous Ort expression pattern in the first-order interneurons of R1-R6 and R7 (Gao, 2009).

    Next, whether the Ort-expressing neurons are required for UV reference and motion detection was examined. ortC1-4-Gal4 or ortC1-3-LexA::VP16 driving a temperature-sensitive allele of shibire, shits1, so as to block synaptic transmission in specific neurons was found to significantly reduce the UV attractiveness at non-permissive, but not permissive, temperatures. This reduction was smaller than that caused by inactivating the R7s alone. These results suggest that Ort-expressing neurons might mediate both UV and green phototaxis, presumably by relaying R7 and R8 channel signals, although the existence of an ort-independent UV-sensing pathway cannot be ruled out. Similarly, inactivating Ort-expressing neurons abolished the flies' ability to detect motion. Thus, it is concluded that Ort-expressing neurons are required for both spectral preference and motion detection (Gao, 2009).

    To identify the Ort-expressing neurons that could be synaptic targets of the R7 and R8 channels, a single-cell mosaic technique based on the flip-out genetic method was employed. In this system, the ortC1-3-Gal4 flies that also carried the transgenes UAS>CD2,y+>CD8-GFP and hs-Flp were used. The flipase activity induced by brief heat-shock at the second- or third-instar larval stages excised the FLP-out cassette in small random populations of cells, thereby allowing Gal4 to drive the expression of the CD8-GFP marker. From over 1000 brain samples, 459 clones of transmedulla neurons, the projection neurons that arborize in the medulla and project axons to the lobula, were examined (see Ort is expressed in subsets of transmedulla neurons). To identify the exact medulla and lobula strata in which these processes extend, expression patterns of a series of known cell-adhesion molecules were screened, and three useful stratum-specific markers, FasIII, Connectin, and Capricious were found. In particular, anti-FasIII immunolabeled medulla and lobula strata of interest and, with MAb24B10 immunolabeling, was used primarily to identify the medulla and lobula strata. Based on the morphologies and stratum-specific locations of the arborization and axon terminals, four types of Ort-expressing projection neurons were assigned to types previously described from Golgi impregnation. These were Tm2, Tm5, Tm9, and Tm20. In addition, the ort-promoter driver labeled, albeit at a lower frequency, centrifugal neurons C2 and T2, and three types of medulla neurons with processes solely in the medulla, Dm8, other amacrine-like and also glia-like cells. All of these cells were identified multiple times in at least two independent ort-Gal4 lines, but given the sampling nature of the single-cell mosaic technique, the possibility cannot be excluded that some very rare Ort-expressing neurons were not detected. The amacrine-like and glia-like cells had not been previously described from Golgi impregnation, suggesting that there are even more classes of medulla cell types than those previously reported (Gao, 2009).

    The Ort-expressing Tm neurons exhibit type-specific patterns of arborization and axon projection (see Ort-expressing Tm neurons receive multi-channel inputs in the medulla and are presynaptic at both the medulla and lobula). Tm5 neurons extend dendrite-like processes in medulla strata M3 and M6, where R8/L3 and R7 axons terminate, respectively, and they project axons to terminate in stratum Lo5 in the lobula. This pattern suggests that they relay information from the R7 and R8 or L3 channels to the lobula. The Tm5 neurons could be readily divided into three subtypes, Tm5a, b, and c, based on their unique dendritic patterns, the spread of their medulla arborization, and their gene expression patterns. Tm5a and Tm5b have medulla arborizations of different sizes and shapes; whereas Tm5c has dendritic processes in M1, in addition to strata M3 and M5, and the axon projects to both the Lo4 and Lo5 strata. The distinct morphology of Tm5c correlates with its unique expression of the vesicular glutamate transporter. Tm9 and Tm20 extend type-specific dendrite-like processes in strata M1-M3 and projected axons to distinct lobula strata. Tm20, like Tm5, projects to Lo5 while Tm9 projects to Lo1, suggesting that Tm9 and Tm20 relay information from R8 and (via lamina neurons) R1-R6, to different strata of the lobula. In medulla strata M1-M3, Tm2 extends dendrite-like processes which did not appear to make significant contacts with R7 or R8 terminals (Gao, 2009).

    To determine if the Ort-expressing Tm neurons indeed receive synaptic input from photoreceptors, serial EM reconstructions were undertaken of Tm9, Tm2, and parts of a single Tm5 cell that resemble Tm5a, as well as the afferent input terminals that innervate the medulla, including R7, R8 and L1-L5 (see also Takemura, 2008). The fine dendritic arbor of Tm20 has so far eluded reconstruction. This study found that Tm9 received direct synaptic contacts from both R8 and L3 and the Tm5 received direct synaptic contacts from R7 and L3. Thus, Tm9 and Tm5 cells were postsynaptic to both the chromatic channels and an achromatic channel. Tm2, by contrast, received synaptic contacts from L2 and L4 but not, despite its Ort expression, R7 or R8. However, the possibility cannot be excluded that Tm2 responds to paracrine release of histamine from the R8 terminal, or an unidentified histamine input in the lobula (Gao, 2009).

    It was reasoned that Ort-expressing neurons might be divided into several groups based on their differential release of other neurotransmitters. To test this possibility, a series of promoter-Gal4 and enhancer trap lines driving the CD8 marker was used to label neurons with glutamatergic, cholinergic, GABAergic, serotonergic and dopaminergic phenotypes in the medulla. To determine whether these neurons also express Ort, and are thus likely to receive histaminergic input, the rCD2::GFP marker was expressed in the same animals using the ortC1-3-LexA::VP16 driver. By overlaying two expression patterns, it was found that many Ort-expressing neurons also expressed cholinergic or glutamatergic markers, while few did so for a GABAergic and none appeared to do so for serotonergic or dopaminergic phenotypes. In particular, it was found that a group of neurons labeled by both the vesicular glutamate transporter (vGlutOK371) and ort-Gal4 drivers extended processes in the M6 stratum where R7 axons terminate, suggesting that R7's target neurons might be glutamatergic (Gao, 2009).

    To identify candidate R7 target neurons, a combinatorial gene expression system, the Split-Gal4 system, was employed to restrict Gal4 activity to glutamatergic Ort-expressing neurons. In this system, ort and vGlut promoters drive expression of the Gal4DBD (Gal4 DNA binding domain-leucine zipper) and dVP16AD (a codon-optimized VP16 trans-activation domain-leucine zipper), respectively. Thus, Gal4 activity was reconstituted only in the neurons that expressed both Ort and vGlut. A dVP16AD enhancer trap vector was used, and it was substituted for the Gal4 enhancer trap in the vGlut locus. The resulting hemidriver, vGlutOK371-dVP16AD, in combination with a general neuronal hemidriver, elav-Gal4DBD, drove expression in a pattern essentially identical to that driven by vGlutOK371-Gal4, indicating that the vGlutOK371-dVP16AD enhancer trap recapitulated the expression pattern of the vGlutOK371-Gal4 driver. The combination of the vGlutOK371-dVP16AD and ortC1-3-Gal4DBD hemidrivers (designated ortC1-3∩vGlut) gave rise to expression in a subset of Ort-expressing neurons in the optic lobe, namely those that express a glutamate phenotype and are thus likely to be glutamatergic. Single-cell mosaic analysis (using hs-Flp and UAS>CD2>mCD8GFP) revealed that the combinatorial ortC1-3∩vGlut driver was expressed in Dm8, Tm5c, and L1 neurons, as well as in the medulla glia-like cells. In contrast, cha∩ortC1-3, the combination of cha-Gal4DBD choline acetyltransferase-Gal4DBD) and ortC1-3-Gal4AD hemidrivers, drove expression in the Ort-expressing neurons that expressed a cholinergic phenotype, including L2, Tm2, Tm9, and Tm20. Notable among these findings, L1 and L2, paired lamina neurons that receive closely matched R1-R6 input in the lamina, express different neurotransmitter phenotypes (L1: glutamate; L2: acetylcholine) (Gao, 2009).

    To determine whether glutamatergic Ort-expressing neurons confer UV preference in flies, whether expressing Ort in these neurons is sufficient to restore normal UV preference in ort mutants was examined. It was found that expressing Ort using the combinatorial ortC1-3∩vGlut driver restored normal UV preference in ort mutants. In contrast, expressing Ort in cholinergic Ort-expressing neurons using the cha∩ortC1-3 driver further reduced UV preference, suggesting that the cholinergic Ort-expressing neurons reduce UV attraction or, more likely, enhance green attraction. Although the cha∩ortC1-3 and ortC1-3∩vGlut drivers were expressed in specific subsets of Ort-expressing neurons in the optic lobe, they showed additional expression outside the visual system, and expressing shits1 with either driver caused non-specific motor defects at the non-permissive temperature. Although it was not possible to test whether the glutamatergic Ort-expressing neurons were required for UV preference, the rescue results indicated that the candidate glutamatergic Ort-expressing neurons, which included Dm8 and Tm5c, were involved in UV preference (Gao, 2009).

    To distinguish whether Dm8 or Tm5c is required for UV preference, the ort promoter was dissected, and three promoter-Gal4 lines were generated, each of which contained one of the three highly conserved regions (C1-C3) of the ort promoter. It was found that the second and the third conserved regions (C2 and C3) gave rise to the expression in two different subsets of Ort-expressing neurons while C1 alone gave no detectable expression. Using single-cell analysis, it was found that ortC2-Gal4 drove expression in Dm8 and L1-L3 but not in any Tm neurons, while ortC3-Gal4 was expressed in L2, Tm2, Tm9, C2, and Mi1 neurons. All these neurons except Mi1 expressed Ort, suggesting that the C2 and C3 fragments of the ort promoter drives expression in distinct subsets of the Ort-expressing neurons, but that the combination of all conserved regions was required to suppress Ort expression in Mi1 (Gao, 2009).

    Next, whether the ortC2 or ortC3 neuron subsets were sufficient and/or required for UV preference was examined. It was found that expressing Ort using the ortC2-Gal4 driver in ort mutants was sufficient to restore UV preference at least up to the wild-type level. Because the lamina neurons L1 and L2 are neither necessary nor sufficient for UV preference, this finding suggested that the Dm8 neurons alone are sufficient to drive a fly's normal preference for UV. Conversely, whether these neurons were required for UV preference was tested using shits1. It was found that flies carrying ortC2->shits1 exhibited strongly attenuated UV preference at the non-permissive, but not permissive, temperatures, indicating that the ortC2 subset is required for normal UV preference. In contrast, restoring the ortC3 subset activity further reduced UV preference, suggesting that the ortC3 subset inhibits UV sensing, or enhances green-sensing pathways. Moreover, blocking the activity of the ortC3 subset using shits1 did not confer a stronger UV preference, suggesting that the ortC3 subset is sufficient but likely not required for phototactic preference to green light (Gao, 2009).

    The preceding evidence indicated that the two lines, ortC2 and ort∩vGlut, together identified the Dm8 neurons both functionally and anatomically as a substrate for UV preference. To test this possibility directly, an ortC2-Gal4DBD hemidriver was generated and combined with the vGlut-dVP16AD hemidriver. It was found that the combinatorial driver ortC2∩vGlut was expressed in most Dm8 neurons as well as in a small number of L1 neurons and glia-like cells. Restoring the expression of Ort in Dm8 in ort or HisCl1 ort double-null mutants completely restored normal UV preference. Conversely, flies carrying ortC2∩vGlut->shits1 exhibited reduced UV preference at the non-permissive, but not permissive, temperature. Thus, the Dm8 are necessary and sufficient for a fly's normal preference for UV (Gao, 2009).

    Finally, using the single-cell mosaic method the morphology of the Dm8 neurons was examined (see Amacrine Dm8 neurons receive direct synaptic input from multiple R7 neurons). In stratum M6 the Dm8 neurons were found to extend web-like processes, which extensively overlap 13-16 R7 terminals. To determine whether Dm8 receives direct synaptic input from R7, an EM marker, HRP-CD2, was examined in the Dm8 neurons using the ortC2-Gal4 driver, and their synaptic structure was examined at the EM level. It was found that most R7 synapses are triads and that Dm8 contributes to at least one of the three postsynaptic elements in essentially all R7 synapses. Cumulatively, Dm8 contributes to ~38% (18 out of 47 identified) of the elements postsynaptic to R7s, suggesting that Dm8 is a major synaptic target for these photoreceptors. In addition, processes of three Dm8 neurons were reconstructed spanning seven medulla columns. It was found that Dm8 processes tiled the M6 stratum with partial overlapping so that each R7 terminal was presynaptic to one or two Dm8 neurons. Examining the presynaptic structures of the Dm8 neurons at EM and light microscopic levels, revealed that the Dm8 neurons were also presynaptic to small-field medulla neurons in stratum M6, including Tm5 and at a few contacts to a cell that resembles Tm9. In summary, the wide-field Dm8 neuron serves as a major target neuron for R7 input and provides output locally in stratum M6 to small-field projection neurons (Gao, 2009).

    Before this study little was known about the synaptic target neurons of the R7 and R8 photoreceptors and the chromatic pathways their connection patterns subserve. This deficit reflected the inability until recently to penetrate the medulla's complexity. This study made use of prior knowledge of neurotransmitters and their receptors in the visual system to design corresponding promoter constructs that identify the first-order interneurons. These neurons were then labeled with genetically encoded markers and their morphology and synaptic connections were examined at the light and electron microscopic levels. Finally, promoter dissection and the Split-Gal4 system were combined with neurotransmitter hemidrivers to target particular neuron subtypes. It is envisioned that the same combinatorial approach can be applied to dissect other complex neural circuits (Gao, 2009).

    This study identified four types of transmedulla neurons, Tm5a/b/c, Tm9, Tm20 and Tm2, that express Ort and are therefore qualified to receive direct input from R7 or R8. These Tm neurons arborize in the medulla and project axons to the lobula, suggesting that they relay spectral information from the medulla to the lobula. Supporting this interpretation, it was found that HA-syt, a presynaptic marker, is indeed localized to their terminals in the lobula. These data support previous suggestions that the lobula plays a key role in processing chromatic information for color vision. Lobula stratum 5 appears most critical for color vision because it receives all three subtypes of Tm5 neurons as well as Tm20. Moreover, it was observed that HA-syt also localized to the dendrite-like processes of all Tm neurons in the proximal medulla, suggesting the presence of presynaptic sites at this level, too. Especially, Tm5a, Tm5b, and Tm20 all extend processes with this presynaptic marker in medulla stratum M8, supporting a previous notion that this stratum might receive chromatic information (Gao, 2009).

    All three subtypes of Tm5 neurons extend processes in medulla strata M6 and M3, suggesting that there they might be postsynaptic to R7 and to R8 or L3. Using serial EM, a Tm5 subtype was partially reconstructed that receives direct synaptic input from both the chromatic UV channel of R7 and the achromatic channel of L3. Serial EM also revealed that Tm9 receives inputs from the chromatic green/blue channel of R8 as well as the achromatic L3 channel. It is tempting to speculate that the Tm9 and Tm5 neurons function as color-opponent neurons by subtracting the L3-mediated luminance signal from the R7/R8 chromatic signal (see Medulla circuits in chromatic information processing). While the detailed neural mechanism must await electrophysiological studies, these anatomical data provide direct evidence that the achromatic and chromatic channels are not segregated. Instead they converge on the first/second-order interneurons, early in the visual pathway (Gao, 2009).

    Using a quantitative spectral preference test, it was determined that in flies the Dm8 neurons are both necessary and sufficient to confer the animals' UV preference. Each Dm8 receives direct synaptic input from ~14 UV-sensing R7s. By pooling multiple R7 inputs, the Dm8 neurons may achieve high UV sensitivity at the cost of spatial resolution. Consistent with this notion, Dm8 is a main postsynaptic partner for R7 terminals: essentially all of R7's presynaptic sites contain at least one Dm8 postsynaptic element. The processes of Dm8 and their synapses with R7s are largely restricted to the medulla stratum M6. The stratum-specific arborization of Dm8 readily explains why R7 photoreceptors that fail to project axons to the M6 stratum are incapable of conferring UV preference (Gao, 2009).

    Dm8 itself has no direct output to higher visual centers in the lobula; instead it is presynaptic to small-field projection neurons, such as Tm5 and possibly Tm9, in the medulla. Thus, Dm8 provides lateral connections linking projection neurons. The morphologies and connections of Dm8 are thus reminiscent of those made by horizontal and amacrine cells in the vertebrate retina. The vertebrate horizontal cells form reciprocal synapses with multiple cones, and in the case where the cones are of different spectral types, the horizontal cells can establish color opponency, as demonstrated in the goldfish retina. Dm8 in Drosophila receives inputs from both Rh3- and Rh4-expressing R7s, but does not provide feedback to photoreceptor terminals, suggesting that Dm8 is unlikely to contribute to color opponency, at least not in a way analogous to vertebrate horizontal cells. Vertebrate amacrine cells have diverse subtypes, which carry out very different functions, including correlating firing among ganglion cells, modulating center-surround balance of the ganglion cells and direction selectivity. The amacrine cells in vertebrate retina receive inputs from bipolar cells and provide the main synaptic input to ganglion cells. It is thus interesting to note that while direct synaptic connections from R7s to Tm5 projection neurons exists, the indirect information flow from R7, to Dm8, and then to Tm5, is both necessary and sufficient to confer UV preference, as suggested by inactivating and restoring experiments. It is hypothesized that the direct and indirect pathways function at different UV intensity levels: Dm8 pools multiple R7 inputs to detect low intensity UV in the presence of high-intensity visible light, while under high intensity UV, Tm5 receives direct input from R7 and mediates true color vision. Further studies using electrophysiology or functional imaging would be required to determine the neural mechanisms of Dm8 (Gao, 2009).

    The spectral preference assay used in this study and others measure relative 'attractiveness' of UV and green light and therefore depends on the visual subsystems sensing UV and green light as well as the interactions between these subsystems. While in simple phototaxis assays, the broad-spectrum and most sensitive photoreceptors, R1-R6, dominate simple phototactic response to both UV and green light, they, as well as their first-order interneurons L1 and L2, appear to play an insignificant or redundant role in spectral preference. Thus, R8 alone, or together with R1-R6, provides the sensory input to promote green phototaxis and/or to antagonize UV attraction. The first-order interneurons that relay R8 input in this context have yet to be identified. While anatomical analysis revealed that Tm9 receives direct synaptic input from R8, the behavioral studies provided only weak and circumstantial evidence for its role in spectral preference. Expressing Ort using the cha∩ortC1-3 or ortC3-Gal4 driver significantly reduced UV preference in ort mutants, and Tm9 is covered by both drivers. Furthermore, inactivating Tm9 using the ortC3 driver and shits1 did not affect UV preference, suggesting that other neurons, such as Tm20, might function redundantly. Verification of these suggestions must await the isolation of Tm9-and Tm20-specific drivers, and the corresponding behavioral studies to assay the effects of perturbing activity in these neurons. It is worth noting that Ort-expressing neurons do not include any Dm8-like wide-field neurons for R8s, and restoring activity in the ortC3 neuron subset is sufficient to confer stronger green preference in ort mutants. It is thus tempting to speculate that Dm8 circuits evolved uniquely to meet the ecological need to detect dim UV against a background of ample visible light (Gao, 2009).

    Differences in neural circuitry guiding behavioral responses to polarized light presented to either the dorsal or ventral retina in Drosophila

    Linearly polarized light serves as an important cue for many animals, providing navigational information, as well as directing them towards food sources and reproduction sites. Many insects detect the celestial polarization pattern, or the linearly polarized reflections off of surfaces, such as water. Much progress has been made towards characterizing both retinal detectors and downstream circuit elements responsible for celestial POL vision in different insect species, yet much less is known about the neural basis of how polarized reflections are detected. Previous studies have established a novel, fully automated behavioral assay for studying the spontaneous orientation response of Drosophila melanogaster populations to linearly polarized light presented to either the dorsal, or the ventral halves of the retina. Separate retinal detectors mediating these responses were detected: the 'Dorsal Rim Area' (DRA), which had long been implicated in celestial POL vision, as well as a previously uncharacterized 'ventral polarization area' (VPA). This study investigated whether DRA and VPA use the same or different downstream circuitry, for mediating spontaneous behavioral responses. Homozygous mutants, or molecular genetic circuit-breaking tools (silencing, as well as rescue of synaptic activity), were used in combination with a behavioral paradigm. It was shown that responses to dorsal versus ventral stimulation involve previously characterized optic lobe neurons, like lamina monopolar cell L2 and medulla cell types Dm8/Tm5c. However, using different experimental conditions, it was shown that important differences exist between the requirement of these cell types downstream of DRA versus VPA. Therefore, while the neural circuits underlying behavioral responses to celestial and reflected polarized light cues share important building blocks, these elements play different functional roles within the network (Velez, 2014).

    Peripheral visual circuits functionally segregate motion and phototaxis behaviors in the fly

    Like the mammalian visual cortex, the fly visual system is organized into retinotopic columns. A widely accepted biophysical model for computing visual motion, the elementary motion detector proposed nearly 50 years ago posits a temporal correlation of spatially separated visual inputs implemented across neighboring retinotopic visual columns. Whereas the inputs are defined, the neural substrate for motion computation remains enigmatic. Indeed, it is not known where in the visual processing hierarchy the computation occurs. Tbis study combined genetic manipulations with a novel high-throughput dynamic behavioral analysis system to dissect visual circuits required for directional optomotor responses. An enhancer trap screen of synapse-inactivated neural circuits revealed one particularly striking phenotype, which is completely insensitive to motion yet displays fully intact fast phototaxis, indicating that these animals are generally capable of seeing and walking but are unable to respond to motion stimuli. The enhancer circuit is localized within the first optic relay and strongly labels the only columnar interneuron known to interact with neighboring columns both in the lamina and medulla, spatial synaptic interactions that correspond with the two dominant axes of elementary motion detectors on the retinal lattice (Zhu, 2009).

    Molecular genetic techniques were used to manipulate neural circuits that mediate two well-known visual behaviors in freely behaving fruit flies: motion-dependent optomotor responses and stationary light-elicited phototaxis responses. To efficiently analyze large numbers of individuals and multiple fly lines, a simple yet robust high-throughput assay was devised that tracks the real-time spatial distribution of up to 100 walking flies responding with either optomotor reflexes to panoramic image movement or fast phototaxis toward a stationary narrow-band light source. To generate apparent motion, an array of LED panels fashioned into a three-sided visual 'hallway' displays a computer-controlled centrifugal-centripetal cycling motion stimulus. Dark stripes against a bright background continuously drift from the center of the hallway toward opposite ends and then switch direction to converge at the center. Flies are contained within a clear acrylic tube in the center of the hallway and tend to distribute themselves evenly along the length of the tube prior to visual stimulation. Wild-type flies move against the direction of image motion such that, in response to centrifugal motion, flies rapidly converge at the center; after a switch to centripetal motion, they segregate equally to the two ends of the hallway arena. Similarly, flies exhibit positive phototaxis and rapidly converge upon a high-intensity LED in the center of the hallway. The spatial distribution of the population over the length of the hallway is calculated online for each video frame in real time and thus provides rapid spatiotemporal measurements of the group walking behavior (Zhu, 2009).

    The walking assay in a combined histological and behavioral screen was used to identify neuronal components that mediate either light-directed or motion-elicited orientation. The UAS/Gal4 system was used to perform a confocal microscopy-based histological screen for Gal4 enhancer trap lines that are expressed in specific groups of neurons of the visual system. These circuits were manipulated with tetanus neurotoxin light chain (TNT), which cleaves neuronal synaptobrevin to suppress synaptic transmission and thus was used to study the behavioral consequences of circuit inactivation (Zhu, 2009).

    In the first visual relay, the lamina, each retinotopic column contains five large monopolar (L) cells: L1-L5. From a screen of more than 300 enhancer trap lines, one (termed Ln-Gal4) showed expression tightly restricted to the lamina. Morphological criteria, analyses of single-cell MARCM clones, and labeling of L4s and L5s with anti-Brain-specific homeobox antibody together reveal that Ln strongly labels L3s and L4s and is expressed at lower level in L2s and L5s. Expression in L1 was undetectable with standard GFP staining but labeled weakly with multiple copies of UAS-mCD8-GFP and UAS-N-sybGFP. The noteworthy feature of this driver is its specificity for lamina cells (Zhu, 2009).

    Inactivating this complement of lamina neurons produced flies that were completely insensitive to visual motion cues in any visual assay. In the walking arena, both control progeny of Ln-Gal4 flies mated with wild-type Canton-S (Ln-Gal4/+), and UAS-TNT/+ flies responded normally to motion signals by converging at the center of the hallway and then dispersing upon motion reversal. Normal optomotor behavior is apparent both in the full spatiotemporal distribution of flies along the hallway and in the temporal dynamics of those flies converging upon the origin of the drifting patterns at the center of the hallway. In contrast, crossing Ln-Gal4 with UAS-TNT eliminated all motion responses. The loss-of-motion responses in the Ln-Gal4/UAS-TNT flies persist under all combinations of spatial, temporal, and contrast conditions tested and also in a standard optomotor flight assay in which these flies failed to respond to either progressive or regressive motionInactivating this complement of lamina neurons produced flies that were completely insensitive to visual motion cues in any visual assay. In the walking arena, both control progeny of Ln-Gal4 flies mated with wild-type Canton-S (Ln-Gal4/+), and UAS-TNT/+ flies responded normally to motion signals by converging at the center of the hallway and then dispersing upon motion reversal. Normal optomotor behavior is apparent both in the full spatiotemporal distribution of flies along the hallway and in the temporal dynamics of those flies converging upon the origin of the drifting patterns at the center of the hallway. On the other hand, crossing Ln-Gal4 with UAS-TNT eliminated all motion responses. The loss-of-motion responses in the Ln-Gal4/UAS-TNT flies persist under all combinations of spatial, temporal, and contrast conditions tested and also in a standard optomotor flight assay in which these flies failed to respond to either progressive or regressive motion (Zhu, 2009).

    Several lines of evidence suggest that, for the Ln-Gal4/UAS-TNT phenotype, signaling is preserved through the L1 pathway and quite possibly through both L1 and L2. First, using TNT to inactivate these cells has little influence on motion responses. Second, phototaxis responses to wavelengths that stimulate photoreceptors R1-R6 and, hence, both L1 and L2 postsynaptically are fully intact in the Ln-Gal4/UAS-TNT flies. Indeed, the phototaxis responses in these animals are significantly stronger than for the intact controls, particularly for green light. Signaling through the lamina would account for the robust phototaxis behavior because without R1-R6 signaling in ninaE flies, phototaxis responses are compromised (Zhu, 2009).

    The opposite influence upon optomotor and phototaxis behavior of inactivating the Ln circuit raises the question of whether this is a property unique to the Ln circuit. Motion and phototaxis responses were compared in the walking assay and it was found that any manipulation to L1 or L2 resulted in reduced phototaxis responses to UV. However, phototaxis responses to green light were not significantly affected. This is in direct contrast to the Ln circuit, which enhances phototaxis responses, particularly for green light (Zhu, 2009).

    The remarkable specificity of the Ln driver for lamina circuits coupled with the stringent behavioral phenotype -- motion blindness and enhanced phototaxis -- suggests that functional segregation of the two behaviors occurs early in the visual pathway. In classical physiological experiments, lamina neurons have been shown to function in tandem with photoreceptors as light-level adaptive high-pass filters. Recent electrophysiological analyses have further suggested that the ON-OFF transient response properties of lamina projection neurons may underlie the remarkably high-performance discrimination of small objects embedded within a background of visual clutter by downstream target-detecting interneurons. The current results provide additional genetic and functional evidence for complex processing within peripheral visual circuits (Zhu, 2009).

    Which neurons implement these computations? TNT does not influence motion sensitivity in L1+L2, suggesting that this circuit does not mediate the motion-blind Ln phenotype. By using a combination of selective genetic inactivation and selective rescue experiments with L1 and L2, Rister (2006) concluded that other lamina neurons (L3 and L5) are neither necessary nor sufficient for motion detection, though both receive either direct or indirect input from the photoreceptors. Due to the conflicting results of L1+L2 manipulation, the current experiments neither confirm nor refute any potential role of L3 and L5, which are labeled by the Ln driver. However, L4 is strongly labeled by Ln and is the only lamina cell that provides regular synaptic connections between neighboring optic columns. Spatiotemporal correlation of light signals from two neighboring visual columns is a hallmark of elementary motion detection. Thus, the hypothesis is compelling that inactivation of L4, in part, underlies the elimination of motion responses in the assays (Zhu, 2009).

    The topology and ultrastructural organization of L4 within the lamina implicates this neuron for elementary motion computations. In Drosophila, physiological and behavioral studies have disclosed that the spatial separation of elementary motion detector (EMD) inputs corresponds to that of the ommatidia lattice, indicating that adjacent visual columns function as paired input arms of the EMD. In addition, a classic study revealed two sets of primary EMDs with approximately equal strength and oriented across the hexagonal array of the compound eye at -30° (-X direction) and +30° (+Y direction) with respect to the equator]. According to SEM reconstructions, there are only two cellular connections between visual columns in the lamina: an irregular amacrine network not involved in motion processing and an ordered L4 network that projects between posteroventral and posterodorsal columns. By aligning the coordinate systems of the functional and anatomical studies, it eas found that the topology of L4 connections precisely matches that of the required interconnection of EMD arrays; through two sister collaterals, each L4 projects to two L2s in neighboring posteroventral (-X) and posterodorsal (+Y) columns (Jay, 2009).

    If L4 receives direct input from L2 in the lamina and if L4 is critical for motion coding, why then does inactivating L2 not produce motion blindness? One possibility is that input to L4 from an amacrine cell [6] could be amplified under conditions in which L2 input is removed. Feedback-dependent mechanisms have been shown to amplify photoreceptor output upon inactivation of postsynaptic histamine channels. The anatomical organization of L4 columnar collaterals is observed within both the lamina and the medulla and is conserved across species. Therefore, another possibility is that synaptic connections to L4 within the medulla, which are presently enigmatic, may carry the requisite inputs (Jay, 2009).

    Definitive characterization of the specific cell circuit that is responsible for the remarkable behavioral phenotype of Ln-Gal4/UAS-TNT flies will require advanced histological reagents and electrophysiological analyses. The results presented in this study lay the groundwork by highlighting lamina and medulla circuits that are vital for conditioning early motion signals. These data emphasize the important role that lamina circuits play in ultimately orchestrating complex visual behaviors such as color vision, phototaxis, and motion-dependent optomotor behaviors (Jay, 2009).

    Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals

    Rival exposure causes Drosophila melanogaster males to prolong mating. Longer mating duration (LMD) may enhance reproductive success, but its underlying mechanism is currently unknown. This study found that LMD is context dependent and can be induced solely via visual stimuli. In addition, it was found that LMD involves neural circuits that are important for visual memory, including central neurons in the ellipsoid body, but not the mushroom bodies or the fan-shaped bodies, and may rely on the rival exposure memory lasting for several hours. LMD is affected by a subset of learning and memory mutants. LMD depends on the circadian clock genes timeless and period, but not Clock or cycle, and persists in many arrhythmic conditions. Moreover, LMD critically depends on a subset of pigment dispersing factor neurons rather than the entire circadian neural circuit. This study thus delineates parts of the molecular and cellular basis for LMD, a plastic social behavior elicited by visual cues (Kim, 2012).

    These findings provide evidence that males retain the memory of rival exposure, based primarily on visual stimuli, for several hours and lengthen mating duration accordingly. Indeed, LMD could be induced by allowing a male to view flies of either sex through a transparent partition, flies of different species or images of themselves in a mirror, indicating that LMD could be generated by visual stimuli without chemical communication. Not only could the LMD defects of per and tim mutants be rescued by the expression of PER or TIM via the nonrhythmic GAL4 driver, expression of PER in PDF neurons was sufficient to restore LMD to per mutants. Moreover, LMD requires electrical activity in lateral neurons, but not some of the dorsal neurons that are important for circadian rhythm. It therefore seems unlikely that circadian rhythm regulation is crucial for LMD. LMD involves the memory of rival exposure that lasts for several hours and is resistant to anesthesia and that it requires the rut function in the ellipsoid body. Finally, it was found that LMD generation depends on the activity of the compound eye, the PDF neurons and a subset of neurons in the ellipsoid body (Kim, 2012).

    Recent studies of social experience-mediated and context-dependent sexual behaviors of the fruit fly implicate chemical communication of males via pheromones as being important. This study found that vision in a social setting is also important for generating LMD. Although a recent report found that males use multiple redundant cues to detect mating rivals, this study found that LMD can be elicited by visual cues because rearing flies in constant darkness eliminated LMD, blind mutants and males with defective vision showed no LMD, and LMD can be generated simply by placing a mirror to allow a singly reared male fruit fly to see his reflection for 5 d. The visual stimulus for LMD likely derives from the red compound eye in motion because LMD can be induced by males of different species or females visible through a transparent film, but not by mutant males without red pigment in their compound eyes (Kim, 2012).

    LMD provides a new method for studying visual memory. To date, learning and memory studies in flies have focused primarily on the memory circuits in mushroom bodies; however, LMD requires a subset of neurons in the ellipsoid body rather than mushroom bodies. The ellipsoid body is the central brain region required for visual learning and memory, whereas mushroom bodies are not required for memory formation in visual learning in a flight simulator. Given that the mating duration assay is simpler than the flight simulator for the investigation of visual memory, it can be useful for large-scale genetic screens to identify mutants with altered visual memory (Kim, 2012).

    Feature detection and orientation tuning in the Drosophila central complex

    Many animals, including insects, are known to use visual landmarks to orient in their environment. In Drosophila, behavioural genetics studies have identified a higher brain structure called the central complex as being required for the fly's innate responses to vertical visual features and its short- and long-term memory for visual patterns. But whether and how neurons of the fly central complex represent visual features are unknown. This study used two-photon calcium imaging in head-fixed walking and flying flies to probe visuomotor responses of ring neurons -- a class of central complex neurons that have been implicated in landmark-driven spatial memory in walking flies and memory for visual patterns in tethered flying flies. Dendrites of ring neurons were shown to be visually responsive and arranged retinotopically. Ring neuron receptive fields comprise both excitatory and inhibitory subfields, resembling those of simple cells in the mammalian primary visual cortex. Ring neurons show strong and, in some cases, direction-selective orientation tuning, with a notable preference for vertically oriented features similar to those that evoke innate responses in flies. Visual responses were diminished during flight, but, in contrast with the hypothesized role of the central complex in the control of locomotion, not modulated during walking. Taken together, these results indicate that ring neurons represent behaviourally relevant visual features in the fly's environment, enabling downstream central complex circuits to produce appropriate motor commands. More broadly, this study opens the door to mechanistic investigations of circuit computations underlying visually guided action selection in the Drosophila central complex (Seelig, 2013).

    Flies display a variety of visual pattern- and position-dependent behaviours, including stripe fixation, short-term orientation memory, pattern learning and place learning. Common to these behaviours is a need to detect and respond to specific features in the insect's visual surroundings. In addition, all these behaviours require the central complex, a deep brain region that has also been implicated in motor control. This study used two-photon calcium imaging in genetically targeted populations of central complex input neurons in behaving flies to investigate their potential visuomotor role. Focus was placed on the dendritic responses of ring neurons -- neurons that connect the lateral triangle (LTR) to the ellipsoid body and have been specifically implicated in visuomotor memory (Seelig, 2013).

    Electron microscopy in the locust has shown that dendrites of ring neuron analogues arborize in specialized structures in the LTR called microglomeruli, where they are contacted by axonal projections from visual areas. Confocal images of the Drosophila LTR labelled with green fluorescent protein (GFP) under the control of a pan-neuronal driver line, R57C10, revealed a similar dense microglomerular substructure in the region (Seelig, 2013).

    It was asked if LTR microglomeruli respond to visual input. Two-photon imaging was used with the calcium indicator GCaMP expressed pan-neuronally to record neural activity in the LTR of head-fixed Drosophila placed at the centre of a visual arena. Flies were presented with small bright vertical bars moving horizontally at different elevations and LTR calcium transients were recorded from several planes of focus on one or both sides of the brain in a single experiment. Calcium transients showed strong temporal correlations at the spatial scale expected of LTR microglomeruli. Visual stimuli evoked robust calcium transients in a subset of microglomeruli, but only when the localized stimuli were in specific spatial locations around the fly. Receptive fields for responsive microglomeruli were computed, and most receptive fields were found to be centered in the ipsilateral visual hemifield. Finally, LTR microglomeruli are clustered retinotopically and principal component analysis based on receptive field centres indicates that they form a spatial map with axes that are almost parallel to the fly's visual field (Seelig, 2013).

    Next the anatomical relationship was examined between the LTR and individual classes of ring neurons that send arborizations to the region. Dendritic arborization patterns were examined of ring neurons targeted by EB1-GAL4, which labels R2 ring neurons required for pattern memory (Pan, 2009), and c232-GAL4, which labels R3/R4d neurons required for spatial memory (Neuser, 2008). In agreement with past anatomical work, different ring neuron classes arborize in specific contiguous parts of the LTR. Each ring neuron in these classes extends dendrites into a single microglomerulus in the LTR, and sends axonal arbors throughout a class-specific ring of the ellipsoid body (Seelig, 2013).

    To understand whether different types of ring neurons have distinctive visual response properties, receptive fields for dendritic microglomeruli of R3/R4d and R2 ring neurons were mapped. Visual responses were found in ~7/40 c232-GAL4-labelled microglomeruli, corresponding to ~7/20 R4d microglomeruli and ~14/20 of R2 microglomeruli labelled by EB1-GAL4. Receptive fields for R2 and R4d neurons cover large parts of the visual field, with highest density near the midline of the ipsilateral visual field. In summary, R4d and R2 microglomeruli appear to have similar visual response properties and overlapping receptive fields, but with peak sensitivity in different parts of the visual field (Seelig, 2013).

    Next the fine structure of microglomerular receptive fields were probed using sparse white noise stimuli. Reverse correlation of microglomerular responses revealed prominent inhibitory subfields in the receptive fields. The spatial scales of receptive field structure that were observe is within the range for visual features that evoke strong innate responses in flies, and that are used for visual pattern learning in tethered flies. To test the validity of these white-noise-based receptive fields, they were used to predict responses to novel bar stimuli. The predicted responses captured much of the temporal and spatial variation in the data, with high correlations between estimated and actual responses (Seelig, 2013).

    Noting that the receptive field structure of ring neuron inputs resembles those of simple cells in mammalian primary visual cortex, it was next asked if these neurons share other response properties. Indeed, when flies were presented with a series of moving oriented bars, strong orientation tuning was found in microglomerular response patterns. As expected for receptive field structures with both excitatory and inhibitory lobes, microglomeruli also showed orientation tuning when presented with bars of opposite contrast, that is, dark bars on a bright background, as are often used in fly behavioural studies. Examining orientation tuning across the population of microglomeruli, a strong preference was observed for vertically oriented bars (Seelig, 2013).

    Next the degree of stereotypy was assessed in response properties of ring neuron dendrites of different flies. Strong correlations across flies were found in receptive field structure for R4d and R2 (Seelig, 2013).

    Ring neurons and the ellipsoid body have often been ascribed a role in complex visuomotor tasks. The possible motor function of ring neurons was examined by assessing potential correlations between neural activity and locomotion in tethered flies walking on an air-supported ball or flying, in darkness or in the presence of a bright stripe moving left or right in front of the fly. Although some R3/R4d neurons did show occasional correlations with locomotion when flies walked in the dark, and responses from visually stimulated animals showed occasional modulation during walking, the changes were within the expected variability of visual responses in stationary flies. Responses were also insensitive to walking direction. Overall, LTR visual responses could be modelled accurately without taking walking state into account. By contrast, responses to visual stimuli were consistently diminished during tethered flight, but showed no obvious correlations with flight direction as assessed by differences in wingbeat amplitude envelope. Thus, ring neuron LTR responses were modulated by motor state, but not in a manner consistent with a direct role in motor coordination, and in a markedly different manner than in the optic lobe (Seelig, 2013).

    Behavioural genetics studies in Drosophila have suggested that the central complex is required for a wide range of important sensorimotor functions. However, in the absence of physiological recordings from the region in flies, it has been challenging to determine its role in the diversity of behaviours that it has been implicated in. This paper has examined the visuomotor responses of ring neurons, which provide input from the LTR to the ellipsoid body of the central complex. Analogous neurons in other insects respond to polarized and unpolarized light, and to mechanosensory stimulation, one of the sensory modalities that this study not explore but which may partly account for unresponsive neurons in this study. It was found that R2 and R4d ring neurons are visually responsive, and these responses are not significantly modulated by walking state. Although visual responses are diminished during flight, they do not vary systematically with wingbeat patterns associated with turns. It is possible that outputs of these ring neurons within the ellipsoid body rings could be more sensitive to motor actions, but the physiological results in their dendrites during behaviour are inconsistent with a major role for these ring neurons in motor coordination in the fly. Further, the high degree of stereotypy that was observe in ring neuron receptive field characteristics across flies indicates that, rather than directly performing motor coordination, these neurons probably provide downstream central complex circuits with similar behaviourally relevant visual feature sets on which to base motor decisions. As a striking example, the strong vertical tuning preference observed in the LTR may partly underlie the tendency of flies to fixate on vertical edges during both flight and walking. Recent work has suggested that vertical stripe fixation during walking largely relies on a hypothesized position system sensitive to local luminance changes that can operate independently of neural circuits involved in optomotor responses to wide-field motion. The response properties of R2 and R4d neurons is consistent with a role in such a position system (Seelig, 2013).

    The retinotopic bias, structure of excitatory and inhibitory subfields, orientation tuning and direction selectivity that were seen are reminiscent of those seen in calcium imaging studies in simple cells in mammalian visual cortex, providing an interesting example of how evolutionarily distant visual systems with different types of eyes nonetheless use similar feature sets to process visual scenes. From Hubel and Wiesel's findings several decades ago to the present, significant progress has been made on identifying neural representations used at different stages in the mammalian visual system. However, mechanisms underlying simple cell responses are not yet fully understood. With its array of genetic tools, Drosophila may allow uncovering how spatiotemporal interactions of excitatory and inhibitory inputs might produce similar orientation tuning and direction selectivity in the LTR. There is considerable evidence for spatially tuned visual responses in the lobula complex and anterior optic tubercle in other insects, suggesting that components of ring neuron input response properties may also arise from selective averaging of weaker and more broadly distributed tuning preferences in such areas (Seelig, 2013).

    Overall, these findings lay the groundwork for future research into how this genetic model organism's small brain uses feature and pattern information for visual orientation and navigation (Seelig, 2013).

    Central complex neurons exhibit behaviorally gated responses to visual motion in Drosophila

    Sensory systems provide abundant information about the environment surrounding an animal, but only a small fraction of that information is relevant for any given task. One example of this requirement for context-dependent filtering of a sensory stream is the role that optic flow plays in guiding locomotion. Flying animals, which do not have access to a direct measure of ground speed, rely on optic flow to regulate their forward velocity. This observation suggests that progressive optic flow, the pattern of front-to-back motion on the retina created by forward motion, should be especially salient to an animal while it is in flight, but less important while it is standing still. This study recorded the activity of cells in the central complex of Drosophila during quiescence and tethered flight using both calcium imaging and whole-cell patch-clamp techniques. A genetically identified set of neurons was identified in the fan-shaped body that are unresponsive to visual motion while the animal is quiescent. During flight their baseline activity increases and they respond to front-to-back motion with changes relative to this baseline. The results provide an example of how nervous systems selectively respond to complex sensory stimuli depending on the current behavioral state of the animal (Weir, 2013).

    Recordings were made of the responses of a class of wide-field fan-shaped body (ExFl1) neurons during flight and quiescence. Using two-photon excitation of the genetically encoded calcium indicator GCaMP3, increased activity was observed in the presynaptic (output) region of these cells with the onset of flight and in response to progressively moving visual patterns during flight. During quiescence the cells were unresponsive to the presented visual stimuli. The cellular responses were tested further using GCaMP5 and whole-cell patch-clamp recordings with patterns of purely progressive or regressive motion, confirming progressive motion sensitivity, and indicating additional prolonged responses after regressive motion (Weir, 2013).

    The observation of flight-dependent visual responses indicates that the ExFl1 neurons must minimally receive input (direct or indirect) from two sets of cells, one sensitive to visual motion and one conveying information about whether or not the animal is flying. Their dendritic branches in the inferior medial protocerebrum and the ventral bodies make it likely that they receive many different types of input. The electrophysiology experiments suggest that single cells might receive input from both visual hemispheres, although they are not conclusive because of high variability in the responses. Unfortunately, little is known of the types of information represented in these two regions and further study will be required to determine the identities of cells presynaptic to the ExFl1 neurons (Weir, 2013).

    There is a larger body of work concerning the fan-shaped body, where ExFl1 cells have output terminals. Although it would be premature to hypothesize the precise identities of downstream neurons, a few general observations are possible. Based on anatomical data, it has been proposed that the fan-shaped body (and the central complex at large) possesses a stereotyped organization composed of large-field input fibers spanning horizontal layers and output fibers arranged in small-field vertical columns (see Young, 2010). The ExFl1 neurons clearly fit in this architecture as an input element. Each ExFl1 neuron spans the entire width of the fan-shaped body. If azimuth is encoded in columnar order, this anatomy would suggest that there should be no retinotopic representation in these cells, which is consistent with current observations (Weir, 2013).

    Similar input cell classes have been anatomically defined for over 20 years. Two types of large-field neurons have been described in the fan-shaped body, those whose fibers traverse the median canal of the ellipsoid body (termed Fm, for median), and those whose fibers take a lateral route, the Fl neurons. The latter (of which the ExFl1 neurons are a subset) are heterogeneous and can be found in all layers of the fan-shaped body. In addition to the ExFl1 neurons, another subset of Fl neurons (ExFl2 cells) has been studied. These cells arborize in layer 5 of the fan shaped body, dorsal to the ExFl1 neurons. Both ExFl1 and ExFl2 neurons have been implicated in visual memory formation (Liu, 2006; Wang, 2008) and it is possible that they convey different aspects of visual information to the fan-shaped body during flight. Phillips-Portillo (2012b) recorded intracellularly from dorsally arborizing fan-shaped body cells in the flesh fly, similar to the ExFl2 neurons in Drosophila (see also Phillips-Portillo, 2012a). These cells fire action potentials at a rate between 5 and 15 Hz in quiescent flies. No responses were observed to a variety of visual and mechanical stimuli, except in one cell, which responded to air puffs and flashes of light in the dorsal field of view. Their activity did not appear to change when animals walked on a Styrofoam ball, although the sample size was limited. Given the current results it is reasonable to propose that the general lack of responsiveness reported in this prior study was due to the inability of the animals to fly during recordings. One cell type possibly postsynaptic to the ExFl1 cells are the so-called pontine neurons that connect the dorsal and ventral layers of a single fan-shaped body column. Phillips-Portillo (2012b) recorded from these cells as well, and observed variable responses to changes in illumination and directional selectivity to moving visual objects in agreement with a role of the fan-shaped body in visual navigation (Weir, 2013).

    Perhaps the most detailed account of the functional anatomy of the central complex has been made in the desert locust Schistocerca gregaria. Researchers working with this species have focused on the central complex as the site of analysis of celestial polarization information. They have identified large numbers of cells that respond to polarized light in the ellipsoid body and the protocerebral bridge, among other regions. However, there is a marked lack of polarization sensitive cells in the fan-shaped body, although this region receives input from visual areas. Variable responses to polarized light from several types of fan-shaped body columnar neurons have been reported. Recently, neurons in the fan-shaped body and other parts of the locust central complex have been reported to respond to translating and expanding visual stimuli, supporting a role in visual control of behavior by this brain region (Rosner, 2013). Candidates for synaptic partners of ExFl1 responsive to flight can be found in the locust literature. The columnar neurons CPU increase activity during flight and conceivably receive input from neurons similar to the ExFl1 neurons (Weir, 2013).

    The current results suggest that the fan-shaped body plays a role in visual processing during flight. One type of visual stimulation that the ExFl1 cells respond to, progressive and regressive optic flow, is experienced when an animal moves forward or backward through the environment. This observation suggests that these neurons might be suited to tasks such as estimating flight speed or measuring forward progress. The aftereffect of increased activity following the cessation of regressive motion is a peculiar feature. Perhaps there is some additional sensitivity to front-to-back acceleration which is triggered by the end of regressive motion, or the cessation of unilateral regressive motion in the receptive field of these cells is itself excitatory. In addition, unilateral and bilateral regressive motion appear to influence the cells differently, suggesting that some comparison between the two sides is taking place. More work is required to explain these phenomena (Weir, 2013).

    The fan-shaped body has been implicated in a variety of behaviors. There have been numerous studies based on genetic intervention in the central complex which are not immediately reconcilable with the current data. Mutations that affect the structure of the central complex result in deficits in walking, expressing tetanus toxin in large-field fan-shaped body neurons results in decreased total walking activity, and the action of various peptides in the fan-shaped body influences locomotor activity. Liu (2006) reported that ExFl1 neurons are 498 involved in memory of an object’s orientation (see also Li, 2009). This discussion focuses on studies that report the electrophysiology of cells, albeit from other species. In Drosophila, more work is necessary before a complete explanation of the role of the fan-shaped body is possible. Research in the cockroach Balberus discoidalis has focused on the function of the central complex during locomotion. Researchers have recorded from fan-shaped body neurons while the animal walks in place on a greased platform or a Styrofoam ball. In this preparation, some neurons in the central complex change their firing rates prior to turns and changes in step frequency (Bender, 2010; Guo, 2013). Of particular interest with respect to this study is an observation of neurons in the central complex that respond to antennal stimulation while the animal is quiescent, but do not respond to the same stimulation while the animal is walking. Bender and co-workers recorded from 15 neurons in the fan shaped body, eight of which were responsive to tactile stimuli while the animal was standing still. Of these, only one responded while the animal was walking in a tethered preparation. Although the effect of active locomotion is opposite to the current findings (reducing instead of educing responses to sensory stimuli), these results lend support to the idea that the fan-shaped body is involved in gating relevant stimuli based on locomotor state. The ability to filter out irrelevant sensory information and focus on behaviorally relevant features is likely a general feature of nervous systems. This study has observed activity-gated visual responses in one small set of neurons in the fan-shaped body. As more cell types are characterized, understanding of the computations performed by the central complex will grow, hopefully leading to an explanation of the role played by this fascinating structure in generating organismal behavior (Weir, 2013).

    Responses of Drosophila giant descending neurons to visual and mechanical stimuli

    In Drosophila, the paired Giant Descending Neurons (GDN), also known as Giant Fibers (GFs), and the paired Giant Antennal Mechanosensory Descending Neurons (GAMDN), are supplied by visual and mechanosensory inputs. Both neurons have the largest cell bodies in the brain and both supply slender axons to the neck connective. The GDN axon thereafter widens to become the largest axon in the thoracic ganglia, supplying information to leg extensor and wing depressor muscles. The GAMDN axon remains slender, interacting with other DN axons medially. GDN and GAMDN dendrites are partitioned to receive inputs from antennal mechanosensory afferents and inputs from the optic lobes. Although GDN anatomy has been well studied in Musca domestica, less is known about Drosophila homologue, including electrophysiological responses to sensory stimuli. This study provides detailed anatomical comparisons of the GDN and the GAMDN, characterizing their sensory inputs. The GDN showed responses to light-ON and light-OFF stimuli, expanding stimuli that result in luminance decrease, mechanical stimulation of the antennae, and combined mechanical and visual stimulation. Ensembles of lobula columnar neurons (type Col A) and mechanosensory antennal afferents are likely responsible for these responses. The reluctance of the GDN to spike in response to stimulation confirms observations of the Musca GDN. That this reluctance may be a unique property of the GDN is suggested by comparisons with the GAMDN, in which action potentials are readily elicited by mechanical and visual stimuli. The results are discussed in the context of descending pathways involved in multimodal integration and escape responses (Mu, 2014).

    Neurodevelopmental origin of behavioral individuality in the Drosophila visual system

    The genome versus experience dichotomy has dominated understanding of behavioral individuality. By contrast, the role of nonheritable noise during brain development in behavioral variation is understudied. Using Drosophila melanogaster, this study demonstrated a link between stochastic variation in brain wiring and behavioral individuality. A visual system circuit called the dorsal cluster neurons (DCN; ~40 clustered neurons located in the dorso-lateral central brain) shows nonheritable, interindividual variation in right/left wiring asymmetry and controls object orientation in freely walking flies. DCN wiring asymmetry instructs an individual's object responses: The greater the asymmetry, the better the individual orients toward a visual object. Silencing DCNs abolishes correlations between anatomy and behavior, whereas inducing DCN asymmetry suffices to improve object responses (Linneweber, 2020).

    Individual variability in morphology is abundant, including among human identical twins and species that reproduce by parthenogenesis. In this regard, the brain is no exception. Examples of individual brain variation include differences of size, weight, and neuroanatomical parcellations of human brains. In invertebrates, where individual neurons can be identified across animals, single neurons show variability in morphology, wiring, synaptic connectivity, and molecular composition across individuals (Linneweber, 2020).

    Similarly, innate behaviors, such as selective attention to stimuli, show individual variation even among genetically identical individuals. The stability of individual differences over time defines behavioral idiosyncrasies as animal individuality. It has been proposed that variability in innate behavior is due to neuromodulation of anatomically hardwired circuits. By contrast, there is evidence for developmental plasticity resulting in a range of possible circuit diagrams among individuals, but whether nonheritable individual anatomical differences in brain wiring can predict distinct behavioral outcomes is unexplored (Linneweber, 2020).

    To test whether stochastic wiring of neural circuits affects behavioral variation, Drosophila contralateral visual interneurons called the dorsal cluster neurons (DCNs) (also known as LC14). DCNs exhibit up to 30% wiring variability of their axonal projections between individuals and between the left and right hemispheres of the same brain. DCN axons innervate two alternative target areas in the fly visual system called the medulla (M-DCNs) and the lobula (L-DCNs). The decision whether any given DCN becomes a M-DCN or L-DCN is determined by an intrinsically stochastic lateral inhibition mechanism mediated by the Notch signaling pathway (Langen, 2013). To test the link between wiring variation and behavioral variation, a visual behavioral assay called Buridan's paradigm was used. In this assay, a fly is placed between two identical high-contrast stripes at 180° from each other in a uniformly illuminated arena. The stripes are unreachable, inducing the fly to walk back and forth between them during the assay (Linneweber, 2020).

    This study reports that flies show behavioral individuality that is nonheritable and is not reduced through inbreeding. The degree in left-right DCN wiring asymmetry in the medulla is a predictor of behavioral performance of individual flies. The more asymmetric the DCN medulla innervation is, the narrower the path a fly walks between the two stripes. DCN activity is necessary for this correlation, and reengineering DCN asymmetry suffices to change an individual's behavior (Linneweber, 2020).

    While analyzing object orientation responses in wild-type Canton S (CS) flies, sex-independent interindividual variability in their trajectories was noted. This study focused on a parameter called absolute stripe deviation (henceforth aSD), measuring the deviation from the narrowest possible path between the stripes. Although males tend to walk narrower paths, the degree of interindividual variation in aSD is the same between males and females. This study therefore continued studies with combined populations (Linneweber, 2020).

    To test whether behavioral variability correlates with genetic diversity, a subset of the Drosophila genomic reference panel (DGRP) was for genetically homogeneous strains with extreme object orientation responses. This identified two strains with opposing behavioral phenotypes: DGRP-639 showed low aSD, whereas DGRP-859 showed high aSD. Similar behavioral differences were found in seven other representative behavioral parameters. However, despite the extreme reduction of genetic diversity, the degree of individual variation in aSD was not reduced. On the contrary, DGRP-639 showed increased behavioral variability, hinting at the nonheritability of this variability (Linneweber, 2020).

    If the genotype of an individual determines its behavior, repeated breeding of parental animals with a specific behavioral trait should select for a specific behavior, creating a behaviorally homogeneous population. Three pairs with the lowest and highest aSD scores, respectively were mated, and object orientation responses were measured in their offspring. No differences were found between the two sets of offspring in aSD scores as well as six other parameters tested. The same was true for the offspring of a single pair with low and high aSD. The same breeding schemes were repeated with the near-isogenic DGRP-639 and DGRP-859 for seven generations. For most parameters, a breeding pair reproduces the full range of variability in the population at every generation (Linneweber, 2020).

    An individual's idiosyncratic behavioral profile may not be heritable either because it is driven by internal-state modulations, or because it is driven by nonheritable developmental mechanisms. To distinguish these possibilities, the same individual CS flies were tested once every other day for 3 days; an individual's behavior was virtually identical over the three trials. Statistical analysis of aSD showed that the individual responses of CS flies on different days were correlated. The same was true for path details like left- or right-shifted angles, distance, full walks, meander, absolute horizon deviation, absolute angle deviation, angle deviation, and center deviation. This analysis was extended over a 4-week period. The object responses of individuals were stable over this extended period. This stability argued against state modulations and in favor of individual properties. Indeed, starvation followed by refeeding over a period of 3 days failed to reduce stability of individual performances despite obvious changes in mean population behavior. Finally, it was asked whether reduced genetic diversity affects behavioral stability. Repeated testing of DGRP-639 and DGRP-859 individual flies was performed; both inbred strains showed temporally stable individual responses (Linneweber, 2020).

    Together, these data show that individual variability in object orientation is a nonheritable, temporally stable trait that is independent of sex, genetic background, and genetic diversity. Where in the brain might such individuality in visual behavior originate (Linneweber, 2020)?

    It has been suggest that object position processing in Drosophila requires qualitative asymmetry of the visual percept of an object. However, direct evidence for this notion is lacking, especially that the sizes of the left and right eyes of the same fly are highly correlated. It has been suggested that binocular interactions, through higher-order commissural visual interneurons, are required for object orientation). Putting the two predictions together this study hypothesized that variation in object orientation responses is regulated by the variation in the asymmetry of a higher-order contralateral visual circuit innervating the frontal visual field. The DCNs match this predicted circuit (Linneweber, 2020 and references therein).

    To obtain a comprehensive description of DCN wiring, this study extended the previous analyses of DCNs that were based on 16 female flies (Langen, 2013), to 103 males and females. The number of DCNs varied from 22 to 68 cells, with a range of 11 to 55 L-DCNs and 6 to 23 M-DCNs. In addition, a distribution of variation was observed in medulla-targeting asymmetry by M-DCNs. The distribution of all DCN asymmetries showed a peak of low asymmetries, although extreme asymmetries were present but rare. Finally, three-dimensional reconstruction showed that M-DCN axons terminate in the posterior medulla, where visual columns from the frontal visual field are located, and the DCN wiring pattern in the medulla does not change in the adult (Linneweber, 2020).

    DCNs represent an ideal candidate for an intrinsically asymmetric population of contralateral higher-order interneurons to mediate object responses. To test this hypothesis, it was first asked whether the DCNs were required for object orientation. Inactivating either all DCNs or only M-DCNs resulted in a strong increase in aSD. Next, the relationship between individual variability in object orientation behavior and individual variability in DCN wiring (N = 103) was queried. Unbiased correlation analysis between 36 behavioral parameters and 37 prominent DCN anatomical features showed that left-right asymmetry in M-DCN innervation correlated with an individual's aSD and other interdependent parameters. Individuals with high M-DCN asymmetry have a low aSD, whereas individuals with symmetric M-DCN have a high aSD. To test if DCN wiring asymmetry is a functional driver of individual object orientation behavior, DCNs were silenced and the analyses was repeated. This abolished the correlation between M-DCN asymmetry and aSD, but not stripe detection per se (Linneweber, 2020).

    The data show that nonheritable developmental variation in DCN wiring asymmetry is necessary for creating variability in object orientation behavior across individuals. It was therefore asked whether the changed object orientation responses in the DGRP strains reflect DCN asymmetry alterations. The low aSD strain DGRP-639 displayed more DCN wiring asymmetry, and the high aSD strain DGRP-859 less DCN wiring asymmetry, consistent with the hypothesis. Next, the DCNs were developmentally rewired either by blocking endocytosis to inhibit developmental signaling among DCNs or by activating the Notch pathway, both in a DCN-specific fashion. This resulted in reduced DCN wiring asymmetry and a correspondingly higher aSD, while preserving the correlation between wiring and behavior. Finally, flies were genetically engineered to generate one-sided DCN clones expressing the neuronal silencer Kir2.1. Animals with asymmetrically silenced clones showed lower aSD scores than controls with unsilenced clones or no clones at all. Together, these data causally link DCN wiring asymmetry to object orientation responses (Linneweber, 2020).

    Finally, to test the hypothesis further, it was asked if generating any asymmetry in visual processing is sufficient to override high stripe deviation. Among 79 CS flies tested, the 20 with the highest aSD indices (>40) were selected, monocular deprivation was performed, and they were tested again. This resulted in a reduction of aSD in these flies, as well as in the entire population (Linneweber, 2020).

    This study has establish a link between variability in the development of the brain and the emergence of individuality of animal behavior. The work shows that intrinsically stochastic mechanisms of brain wiring give rise to intraindividual variation of left-right asymmetry in the innervation of the fly visual areas, which explains the individuality of behavioral differences in object responses. The amenability of the relatively complex Drosophila brain to multiscale analysis, from the molecular to the behavioral, at single-animal resolution makes it a model for understanding the emergence of individuality at each of these scales. It is speculated that similar mechanisms and consequences will hold true in other species, including humans (Linneweber, 2020).

    Previous work in Drosophila visual behavioral neuroscience led to the proposal that asymmetry in visual information processing influences object responses. Where such functional asymmetry lay and how it might arise has, until now, remained unclear. Independently, the study of object responses in motion-blind mutants led others to propose a hypothetical contralateral circuit dedicated to object responses in the frontal visual field. The discovery that DCN asymmetry drives object orientation responses in individuals is an elegant solution combining both predictions: a contralateral asymmetric visual circuit that regulates object orientation in the frontal visual field. Future work will reveal the exact physiological consequences of morphological asymmetry, such as whether wiring asymmetry induces timing differences as in auditory navigation or whether the absolute differences are simply summed up (Linneweber, 2020).

    This work provides evidence for the generation of multiple brain and behavior phenotypes from the same genotype via developmental stochasticity and noise. This can serve as a robustness factor for both the individual and the population by increasing the chances of survival of any given genome in case of strong selection pressure (Linneweber, 2020).

    Development of the anterior visual input pathway to the Drosophila central complex

    The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. This paper analyzes the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6-P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1-4, form the posterior EB primordium. Starting out as an integral part of the fan-shaped body (FB) primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. This paper documents the extension of neuropil glia around the nascent EB and BU and analyzes the relationship of primary and secondary neurons of the AVP lineages (Lovick, 2017).

    The central brain of Drosophila is formed by a relatively small number of fixed neural lineages that are produced by genetically unique, stem cell-like neuroblasts. Neural lineages represent genetic modules, as well as structural modules. In the embryo, each neuroblast is defined by the dynamic expression of a specific set of transcription factors. The genetic address provided by these factors plays an essential role in shaping the morphology and function of the corresponding lineage. Lineages also form structural modules, in that neurons of a lineage form compact clusters of cells and emit axons that bundle into one or two coherent fascicles, the primary and secondary lineage axon tracts. Furthermore, arborizations of a given lineage are spatially confined to one or a few individual neuropil compartment(s). Visualizing lineages by clonal analysis has provided a map of the 'macrocircuitry' of the Drosophila brain (Lovick, 2017).

    A recent study has shown that the neural circuit providing visual input to the central complex, the anterior visual pathway (AVP), is formed by three lineages, DALv2, DALcl1, and DALcl2 (Omoto, 2017; see Figure 1 Anterior visual pathway of the Drosophila brain). Larvally-born (secondary) neurons of DALv2 are confined to the ellipsoid body and its input compartment, the bulb (BU). They form the so-called ring (R) neurons of the ellipsoid body, which play a pivotal role in visual memory and complex visually guided behaviors, as well as multiple other functions, controlled by the central complex. Different R-neuron classes of the ellipsoid body are defined by their central-peripheral position in the ellipsoid body, which in turn is correlated to dendritic location in the bulb. Most easily observed in the horizontal plane of the EB, five discrete domains of the ellipsoid body (EBa/oc/ic/op/ip) can be delineated based on the density of anti-DN-cadherin staining, a global marker of neuropil (Omoto, 2017). Ring neurons typically respect boundaries of these domains, generating a basis by which they can be more definitively classified. Most outer ring neurons with axons confined to the outer central (Roc) and anterior domain (Ra) of the EB have dendritic endings in the superior bulb. Some outer central ring neurons innervate the anterior bulb. The inner ring neurons that arborize in the inner central and inner posterior EB domain (Ric, Rip) possess dendrites in the inferior bulb (Lovick, 2017).

    Input to the bulb (BU) originates in the anterior optic tubercle (AOTU), which in turn is a recipient of visual interneurons from the medulla. Two hemilineages (sets of neurons derived from the unequal division of the ganglion mother cells), DALcl1d and DALcl2d (Omoto, 2017), form a topographically ordered projection from AOTU to BU that respects the boundaries set by the subclasses of DALv2 ring neurons. DALcl1 and DALcl2 are two neighboring lineages with cell bodies in the dorso-anterior cortex. Both include a hemilineage whose axon tract passes dorsal of the peduncle (DALcl1/DALcl2d), and another one passing ventrally (DALcl1v/DALcl2v). Only the dorsal hemilineages contribute to the AVP. DALcl1d neurons connect the lateral domain of the AOTU (AOTUl, AOTUil) with the superior and anterior BU (TuBus; TUBua). DALcl2d neurons connect the intermediate medial domain of the AOTU (AOTUi,) to the inferior BU (TuBui). The AOTUl and AOTUi receive input from the medulla of the optic lobe via different populations of multi-columnar medullary projection neurons (MeTu). Functional studies of DALcl1/2 input to the bulb demonstrated that the spatially and developmentally discrete subpopulations of neurons targeting the superior bulb have properties that fundamentally differ from the inferior bulb pathway: the former is activated by small ipsilateral stimuli and projects in a precise retinotopic manner onto the bulb; the latter are inhibited by ipsilateral stimuli, and are activated by contralateral stimuli distributed widely over the visual field (Omoto, 2017; Lovick, 2017 and references therein).

    Similar to the AVP itself, output pathways from the ellipsoid body are also structured around lineages. For example, PB-EB-gall neurons are sublineages of four large (type II) lineages. These include DM1/DPMm1, DM2/DPMpm1, DM3/DPMpm2, DM4/CM4, from here on onward called DM1-DM4 in the text. PB-EB-gall neurons form a topographically highly ordered pathway that interconnects small segments of the protocerebral bridge (PB; the posterior-most compartment of the central complex) with the ellipsoid body and the lateral accessory lobe, a known premotor area of the insect brain (Lovick, 2017).

    With the properties summarized above, the AVP exemplifies a circuit where structurally and functionally discrete classes of neurons represent developmental units, that is, components of a small number of lineages. This paper investigates the development of the AVP. Previous works had already shed light on developmental and evolutionary aspects of the central complex; however, a detailed analysis of the sequential steps leading up to the formation of the ellipsoid body and its input pathway had not been carried out. In this work, using markers expressed throughout development, it was possible to follow the first appearance of the different elements of the AVP in the larva, and map the larval primordia of this pathway. Like larval primordia of adult neuropil compartments in general, primordia of the AVP are formed by the undifferentiated axon tracts of lineages DALv2, DALcl1/2, and the posterior type II lineages, which represent dense bundles with filopodia, lacking the expression of synaptic markers that are characteristic for differentiated neuropil. During the first 48 hours of metamorphosis, these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. Of particular interest was the formation of the EB, which consists of two very different populations of neurons: wide-field R neurons of lineage DALv2, located in the anterior brain, and small-field (columnar) neurons of the DM1-4 lineages, located in the posterior brain. These two groups initially form two separate primordia which later merge into the EB. Finally, this study has provided a first description of the projection of the primary neurons of lineages DALv2 and DALcl1/2. Given that a central complex and AVP, in the anatomical sense, are not yet formed in the larval brain, it was asked what compartments are innervated by the primary neurons of these lineages, and how they relate to the adult central complex. The data provide a foundation for future studies addressing the genetic and developmental mechanisms by which complex, homotopically ordered pathways are controlled (Lovick, 2017).

    Auditory activity is diverse and widespread throughout the central brain of Drosophila

    Sensory pathways are typically studied by starting at receptor neurons and following postsynaptic neurons into the brain. However, this leads to a bias in analyses of activity toward the earliest layers of processing. This paper presents new methods for volumetric neural imaging with precise across-brain registration to characterize auditory activity throughout the entire central brain of Drosophila and make comparisons across trials, individuals and sexes. It was discovered that auditory activity is present in most central brain regions and in neurons responsive to other modalities. Auditory responses are temporally diverse, but the majority of activity is tuned to courtship song features. Auditory responses are stereotyped across trials and animals in early mechanosensory regions, becoming more variable at higher layers of the putative pathway, and this variability is largely independent of ongoing movements. This study highlights the power of using an unbiased, brain-wide approach for mapping the functional organization of sensory activity (Pacheco, 2021).

    Flies detect sound using a feathery appendage of the antenna, the arista, which vibrates in response to near-field sounds. Antennal displacements activate mechanosensory Johnston's organ neurons (JONs) housed within the antenna. Three major populations of JONs (A, B and D) respond to vibratory stimuli at frequencies found in natural courtship song. These neurons project to distinct areas of the antennal mechanosensory and motor center (AMMC) in the central brain. Recent studies suggest that the auditory pathway continues from the AMMC to the wedge (WED), then to the ventrolateral protocerebrum (VLP) and to the lateral protocerebral complex (LPC). However, knowledge of the fly auditory pathway remains incomplete, and the functional organization of regions downstream of the AMMC and WED are largely unexplored. Moreover, nearly all studies of auditory coding in Drosophila have been performed using female brains, even though both males and females process courtship song information (Pacheco, 2021).

    To address these issues, methods were developed to investigate the representation of behaviorally relevant auditory signals throughout the central brain of Drosophila and to make comparisons across animals. Two-photon microscopy was used to sequentially target the entirety of the Drosophila central brain in vivo, combined with fully automated segmentation of regions of interest (ROIs). In contrast to recent brain-wide imaging studies of Drosophila, temporal speed for was traded off for enhanced spatial resolution. Imaging at high spatial resolution facilitates automated ROI segmentation, with each ROI covering subneuropil structures, including cell bodies and neurites. ROIs were accurately registered into an in vivo template brain to compare activity across trials, individuals and sexes, and to build comprehensive maps of auditory activity throughout the central brain. The results reveal that the representation of auditory signals is broadly distributed throughout 33 out of 36 major brain regions, including in regions known to process other sensory modalities, such as all levels of the olfactory pathway, or to drive various motor behaviors, such as the central complex. The representation of auditory stimuli is diverse across brain regions, but focused on conspecific features of courtship song. Auditory activity is more stereotyped (across trials and individuals) at early stages of the putative mechanosensory pathway, becoming more variable and more selective for particular aspects of the courtship song at higher stages. This variability cannot be explained by simultaneous measurements of ongoing fly behavior. Meanwhile, auditory maps are largely similar between male and female brains, despite extensive sexual dimorphisms in neuronal number and morphology. These findings provide the first brain-wide description of sensory processing and feature tuning in Drosophila (Pacheco, 2021).

    Sensory systems are typically studied starting from the periphery and continuing to downstream partners guided by anatomy. This has limited the understanding of sensory processing to early stages of a given sensory pathway. This study used a brain-wide imaging method to unbiasedly screen for auditory responses beyond the periphery and, via precise registration of recorded activity, to compare auditory representations across brain regions, individuals and sexes. Auditory activity was found to be widespread, extending well beyond the canonical mechanosensory pathway, and is present in brain regions and tracts known to process other sensory modalities (that is, olfaction and vision) or to drive motor behaviors. The representation of auditory stimuli diversified, in terms of both temporal responses to stimuli and tuning for stimulus features, from the AMMC to later stages of the putative pathway, becoming more selective for particular aspects of courtship song (that is, sine or pulse song, and their characteristic spectrotemporal patterns). Auditory representations were more stereotypic across trials and individuals in early stages of mechanosensory processing, and more variable at later stages. By recording neural activity in behaving flies, this study found that fly movements accounted for only a small fraction of the variance in neural activity, which suggests that across-trial auditory response variability stems from other sources. These results have important implications for how the brain processes auditory information to extract salient features and guide behavior (Pacheco, 2021).

    Understanding of the Drosophila auditory circuit thus far has been built up from targeted studies of neural cell types that innervate particular brain regions close to the auditory periphery. Altogether, these studies have delineated a pathway that starts in the Johnston's organ and extends from the AMMC to the WED, the VLP and the LPC. By imaging pan-neuronally, widespread auditory responses that spanned brain regions beyond the canonical pathway, which suggests that auditory processing was found to be more distributed. However, for neuropil signals, it was challenging to determine the number of neurons that contribute to the described ROI responses. Although the diverse set of temporal and tuning types sampled per neuropil suggests that many neurons per neuropil, restricting GCaMP to spatially restricted genetic enhancer lines will assist with linking broad functional maps with the cell types constituting them (Pacheco, 2021).

    Findings of widespread auditory activity are likely not unique to audition. So far, in adult Drosophila, only taste processing has been broadly surveyed. While that study did not map activity onto neuropils and tracts, nor did it make comparisons across individuals, it suggested that taste processing was distributed throughout the brain. Similarly, in vertebrates, widespread responses to visual and nociceptive stimuli have been observed throughout the brain. The findings are consistent with anatomical studies that found connections from the AMMC and the WED to several other brain regions, and with the analysis of the hemibrain connectome. While it is not yet known what role this widespread auditory activity plays in behavior, this study shows that ROIs that respond to auditory stimuli do so with mostly excitatory (depolarizing) responses and that activity throughout the brain is predominantly tuned to features of the courtship song. During courtship, flies evaluate multiple sensory cues (olfactory, auditory, gustatory and visual) to inform mating decisions and to modulate their mating drive. Although integration of multiple sensory modalities has been described in higher-order brain regions, the results suggest that song representations are integrated with olfactory and visual information at earlier stages. In addition, song information may modulate the processing of non-courtship stimuli. Song representations in the MB may be useful for learning associations between song and general olfactory, gustatory or visual cues, while diverse auditory activity throughout all regions of the LH may indicate an interaction between song processing and innate olfactory behaviors. Finally, auditory activity was found in brain regions involved in locomotion and navigation (the central and lateral complex, and the superior and ventromedial neuropils). Auditory activity in these regions is diverse, which suggests that pre-motor circuits receive information about courtship song patterns and could therefore underlie stimulus-specific locomotor responses (Pacheco, 2021).

    D. melanogaster songs are composed of pulses and sines that differ in their spectral and temporal properties; however, it is unclear how and where selectivity for the different song modes arises in the brain. Since neurons in the LPC are tuned for pulse song across all time scales that define that mode of song, neurons upstream must carry the relevant information to generate such tuning. This study found many ROIs that are selective for either sine or pulse stimuli throughout the entire central brain. A more detailed systematic examination of tuning in the neuropils that carry the most auditory activity (the AMMC, the SAD, the WED, the AVLP and PVLP, the PLP and the LH) revealed that most ROIs are tuned to either pulse or sine features, with few ROIs possessing intermediate tuning. This suggests that sine and pulse information splits early in the pathway. It was also found that sine-selective responses dominate throughout the brain. Although some of this selectivity may simply reflect preference for continuous versus pulsatile stimuli, this investigation of feature tuning revealed that many of these ROIs preferred frequencies that are specifically present in courtship songs. Previous studies indicated that pulse song is more important for mating decisions, with sine song purported to play a role in only priming females. However the results, in combination with the fact that males spend a greater proportion of time in courtship singing sine versus pulse song, suggest a need for reevaluation of the importance and role of sine song in mating decisions. This study therefore lays the foundation for exploring how song selectivity arises in the brain (Pacheco, 2021).

    The results also revealed that early mechanosensory brain areas contain ROIs with less variable auditory activity across trials and animals. The results for across-animal variability have parallels to the Drosophila olfactory pathway, whereby third-order MB neurons are not stereotyped, while presynaptic neurons in the AL, the PNs, are. Similarly, a lack of stereotypy beyond early mechanosensory brain areas may reflect stochasticity in synaptic wiring. The amount of variation observed in some brain areas was large, and follow-up experiments with sparser driver lines will be needed to validate whether what is reported here applies to variation across individual identifiable neurons (Pacheco, 2021).

    A wide range of across-trial variability was observed throughout neuropils with auditory activity. Imaging from a subset of brain regions in behaving flies revealed that trial-to-trial variance in auditory responses is not explained by spontaneous movements, which suggests that variance is driven by internal dynamics. This result differs from recent findings in the mouse brain, which showed that a large fraction of activity in sensory cortices corresponds to non-task-related or spontaneous movements. This may indicate an important difference between invertebrate and vertebrate brains and the degree to which ongoing movements shape activity across different brains. However, it should be pointed out that while motor activity is known to affect sensory activity in flies, this modulation is tied to movements that are informative for either optomotor responses or steering. In these experiments, although flies walked abundantly, they did not produce reliable responses to auditory stimuli, although playback of the same auditory stimuli can reliably change walking speed in freely behaving flies. Adjusting the paradigm to drive such responses might uncover behavioral modulation of auditory activity. Alternatively, behavioral modulation of auditory responses may occur primarily in motor areas, such as the central complex, or areas containing projections of descending neurons. Further dissection of the sources of this variability would require the simultaneous capture of more brain activity in behaving animals while not significantly compromising spatial resolution (Pacheco, 2021).

    This paper provides tools for characterizing sensory activity registered in common atlas coordinates for comparisons across trials, individuals and sexes. By producing maps for additional modalities and stimulus combinations and by combining these maps with information on connectivity between and within brain regions, the logic of how the brain represents the myriad stimuli and their combinations present in the world should emerge (Pacheco, 2021).

    Boyan, G., Williams, L., Ehrhardt, E. (2023). Central projections from Johnston's organ in the locust: Axogenesis and brain neuroarchitecture. Dev Genes Evol, 233(2):147-159 PubMed ID: 37695323

    Central projections from Johnston's organ in the locust: Axogenesis and brain neuroarchitecture

    Johnston's organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the locust, this pathway is already present at mid-embryogenesis but the process of fasciculation involved in its construction has not been investigated. Terminal projections into the fine neuropilar organization of the brain also remain unresolved, information essential not only for understanding the neural circuitry mediating Jo-mediated behavior but also for providing comparative data offering insights into its evolution. In this study, neuron-specific, axon-specific, and epithelial domain labels were employed to show that the pathway to the brain of the locust is built in a stepwise manner during early embryogenesis as processes from Jo cell clusters in the pedicel fasciculate first with one another, and then with the two tracts constituting the pioneer axon scaffold of the antenna. A comparison of fasciculation patterns confirms that projections from cell clusters of Jo stereotypically associate with only one axon tract according to their location in the pedicellar epithelium, consistent with a topographic plan. At the molecular level, all neuronal elements of the Jo pathway to the brain express the lipocalin Lazarillo, a cell surface epitope that regulates axogenesis in the primary axon scaffold itself, and putatively during fasciculation of the Jo projections to the brain. Central projections from Jo first contact the primary axon scaffold of the deutocerebral brain at mid-embryogenesis, and in the adult traverse mechanosensory/motor neuropils similar to those in Drosophila. These axons then terminate among protocerebral commissures containing premotor interneurons known to regulate flight behavior (Boyan, 2023).


    Boyan, G., Williams, L., Ehrhardt, E. (2023). Central projections from Johnston's organ in the locust: Axogenesis and brain neuroarchitecture. Dev Genes Evol, 233(2):147-159 PubMed ID: 37695323

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    Glia and axonogenesis

    Separate sections of The Interactive Fly group genes according to their involvement in glia morphogenesis and axonogenesis.

    genes expressed in brain morphogenesis

    Genes involved in organ development

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