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).

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).

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).

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).

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., Tm_LM7 and Pm_LM7) 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, Tm_LM7 and Tm_LM8 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).

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/ort^US2515 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/ort^US2515 mutants (as well as other allelic combinations, ort^P306/ort^US2515 and ort¹/ort^P306) 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 (Attr_UV/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 ort^P306 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).

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).

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).

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).

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 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).

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).

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).