Development of the lamina visual center of the brain

A major part of the adult insect protocerebrum is the optic lobe, consisting of the first, second and third optic ganglion (lamina, medulla and lobula/lobula plate, respectively). In Dipterans, the optic lobe develops from an epithelial placode which invaginates from the procephalic ectoderm, posteriorly adjacent to (and partially overlapping with) the posterior protocerebral neuroblasts. The optic lobe placode (not to be confused with the eye-antennal disc which has an independent origin) invaginates during stages 12 and 13. Its cells constrict apically, leading to the formation of a vertically elongated groove in the center of the optic lobe placode. By the end of stage 12 the optic lobe invagination forms a deep pouch containing about 85 cells. During the invagination process, the placode adopts a V-like shape, with a conspicous anterior and posterior lip. It is thought that the posterior lip gives rise to the outer optic anlage, which in turn produces the lamina and medulla, and that the anterior lip forms the inner optic anlage which mainly produces the lobula/lobula plate complex (Younossi-Hartenstein, 1996 and references).

The optic lobes of the adult brain are derived from neuroblasts organized during the third instar larvae into two columnar epithelia: the inner proliferation center (ipc) and the outer proliferation center (opc).

First instar: A total of 30 to 40 precursor cells of the optic anlagen are found superficially in the lateral cell body layer of each brain hemisphere at the time of hatching (the transition from embryonic development to the first larval stage). These cells differ from the remaining cells of the hemisphere due to their somewhat smaller size. Within the early first instar, labelled nuclei appear in these smaller cells. The cells become larger, ellipsoid and epithelially arranged. From the second half of the first instar onwards two different epithelia can be distinguished; these develop into the opc and ipc.

Second instar: No obvious pattern of labeling is detectable until the end of the second instar, when the production of postmitotic neurons begins. At the end of the second instar, there are about 700 neuroblasts in the opc and about 400 neuroblasts in the ipc. Subsequently, a proliferation zone is formed at the medial edge of the opc. Mitotic figures and some small ganglion mother cells can be distinguished adjacent to the neuroblasts. The number of cells produced by this proliferation zone amounts to approximately 40,000, consisting of the medulla, the outermost cell layer of the optic lobe (Hofbauer, 1991).

Third instar: During the middle of the third instar, a second proliferation zone (still part of the opc) develops at the lateral rim of the opc. This zone consists of neuroblasts that have become separated from the main anlage by a deep furrow. Ganglion mother cells and ganglion cells are produced at the inner edge of this crescent. However, this zone yields many fewer cells than found in the medulla anlagen (about 6000 at 25 hours after puparium formation). These cells form the lamina (Hofbauer, 1991). Since mitotically active lamina precursor cells, which normally produce lamina neurons, are absent in mutants that lack retinal innervation, it is concluded that the arrival of photoreceptor axons in the larval brain initiates cell division to produce lamina neurons (Selleck, 1991).

Two different populations of ganglion cells originate from the lateral proliferation zone of the ipc. Most of the cells differentiate to become the cell body layer of the lobula plate. The other cells participate in the formation of the inner medulla neuropil. About 15000 cells are generated from the lateral proliferation zone. An additional small dorsal proliferation zone develops from the ipc and these cells become part of the lobula cell body layer (Hofbauer, 1991).

Ganglion cells begin to grow axons shortly after their final mitosis: corresponding to the gradient of cell proliferation, there is also a gradient of differentiation in each developing neuropil. The axons of the lamina cells grow centrally, forming a fine fiber sheet at the inner margin of the lamina cell body area at right angles to the medulla neuropil. When differentiation of the lamina starts during the middle of the third instar, the youngest part of the lamina neuropil is in contact with the youngest, most lateral part of the medulla neuropil. The cells of the lobula complex send their axons centrally and form a neuropil opposite and almost parallel to the medulla neuropil. Lobula and lobula plate will develop from this fiber mass. The first fibers connecting the different visual neuropils appear very early, at a time when only the minor part of the ganglion cells are postmitotic. Photoreceptor cell axons start growing into the brain during the middle of the third instar, beginning at the posterior edge of the developing eye and progressing towards the anterior. At the same time, the proliferation of lamina ganglion cell progenitors begins and the newly generated lamina cells become connected with newly ingrowing photoreceptor cells (Hofbauer, 1991).

The lamina is a ganglion layer of the visual center of the brain that processes information received from R1-R6 photoreceptor neurons located in the ommatidia of compound eyes. The R1-R6 photoreceptors send their axons to the lamina, where they are distributed in a "neurocrystalline" array of postsynaptic "cartridge" units. Each cartidge unit contains a set of R1-R6 axons and five lamina neurons and is associated with glia of four defined types.

In eyeless mutants, the lamina is completely absent, and the other visual ganglia are reduced in size. The dependence of lamina development on innervation from the compound eye is due to direct coupling of neurogenesis to the arrival of retinal axons from the eye. As photoreceptor cell differentiation and ommatidial assembly sweep across the eye disc epithelium, the arrival of axons from each new ommatidial photoreceptor cell cluster triggers a corresponding group of lamina precursor cells (LPC) to undergo a final cell division and commence differentiation. In animals lacking retinal innervation, LPCs fail to undergo their terminal division and remain in G1 arrest until eliminated by cell death. Direct contact with retinal axons triggers the transition of G1-phase LPCs into S Phase.

Morphogenesis in the lamina is structured in a linear fashion, taking place sequentially as axonic innervation arrives in the brain. Morphogenesis takes place along a furrow that moves across the developing lamina, in a manner directly related to the progression of the morphogenetic furrow of the eye optic disc. Arrival of axons trigger LPCs to divide at the posterior margin of the lamina furrow, initiating the events that give rise to lamina neurons. In the lamina, the LPC progeny are organized into columns in precise register with the axons that regulate their differentiation. LPC progeny and glia of several types assume stereotyped positions in the lateral medial axis. The glia include medulla, marginal, epithelial and stellate types.

The secreted protein Hedgehog has been identified as the signal transmitted along retinal axons which serves as the inductive signal triggering neurogenesis in the lamina. patched is expressed in two classes of lamina glia and in subretinal glia cells in the optic stalk and the eye disc, cell populations that are in close proximity to retinal axons. Ectopic hh expression in the brains of eyeless flies induces lamina differentiation, but is not sufficient to induce Elav, a late marker. This suggests that HH alone is not sufficient for the later events of lamina development that include the specification of LPC progeny as lamina neurons (Huang, 1996).

The target of HH in the developing visual system is wingless, which in turn targets decapentaplegic and Distal-less. The lamina neurons and the cortical neurons that contribute axons to the medulla neuropil derive from a neuroblast population (OPC or outer proliferation center) that divides throughout most of larval development. Although cells expressing wg constitute only a small fraction of the OPC, the inactivation of wg at early times results in the later absence of nearly the entire target structure. Inactivation of wg at later times results in the formation of pregressively more complete central target structures. The expression of wg in the developing visual system is consistent with a role in organizing the dorsoventral axis. wg expression begins in a single ventral cell at approximately 10 hr of larval develpment. About 6 hr later, wg expression begins in a single dorsal cell. For the remaining stages of larval development wg is expressed in two OPC domains that define the dorsal and ventral termini of the developing target regions. These observations suggest that wg has a nonautonomous role in organizing the target region of retinal axons. The fact that early wg expression occurs prior to axon ingrowth, indicates that HH is required for the maintenance of wg expression and not for its initiation (Kaphingst, 1994).

Wingless regulates the onset and maintenance of dpp expression. Approximately 14 hr after the onset of wg expression, dpp expression begins in single cell domains immediately adjacent to the wg-expressing cells, and is maintained throughout larval development as these cell populations divide up to and including the period of retinal axon ingrowth. In dpp mutants many OPC progeny fail to down-regulate the expression of the cell adhesion molecule fasII, fail to express neuron markers, and fail to contribute axons to the medulla neuropil (Kaphingst, 1994).

Distal-less expression is found in wg-expressing cells adjacent to the dpp domains. dll expression is significantly greater in the dorsal domain. The involvement of dll in neurogenesis in Drosophila has yet to be documented (Kaphingst, 1994).

Minibrain is a serine/threonine kinase involved in proliferation of cells of the opc. When the development of optic lobes in minibrain mutants is compared with that of wild type, it is found that the opcsin mutants attain an irregular structure. The opcs are distinguishable in third instar larvae, appearing in tightly packed, ribbon-like layers of cells, demarcated from the hemisphere. The proliferating neuroblasts appear as a very regular dome-like pattern in wild-type brain. One thick and two thin ribbons of bromodeoxuridine (BrdU) labeled cells (indicating cells that have gone through the DNA synthetic S phase) can be distinguished in the distal brain hemispheres. This regular spatial distribution of BrdU labeled neuroblasts is disturbed in the opcs of mnb mutants. In strong alleles, this ribbon of opc neuroblasts is condensed, and the BrdU-labelled nuclei are not distributed evenly. The regular structure of the thin ribbons disappears, resulting in a scattered distribution of labeled nuclei in these areas. It is important to note that in most cases the altered opc structure does not apparently change the number of labeled cells in comparison with wild type. Nevertheless, abnormally large cells with dark nuclei (probably degenerating cells) are frequently seen near to neuroblast-like cells. In comparison with wild type, mutants are missing a clear demarcation between neuroblasts and gangion cell bodies. In contrast to mutant opcs, no detectable structural alteration is seen in mutant ipcs. From observations of pupal brains, it is concluded that the minibrain phenotype is primarily caused by the inability of mutants to generate a sufficient number of optic lobe and central brain neurons during postembryonic development (Tejedor, 1995).

Cell proliferation within the optic lobe anlagen is dependent on ecdysteroids during metamorphosis of the moth Manduca sexta. Cultured tissues were used to show that ecdysteroids must be maintained above a sharp threshold concentration to sustain proliferation. Proliferation can be turned on and off repeatedly simply by shifting the ecdysteroid concentration to levels above or below this threshold. In subthreshold hormone, cells arrest in the G2 phase of the cell cycle. Ecdysteroid control of proliferation is distinguished from differentiative and maturational responses to ecdysteroids by requiring tonic exposure to the hormone and lower levels of 20-hydroxyecdysone, and by being sensitive to either 20-hydroxyecdysone or its precursor, ecdysone. These characteristics allow optic lobe development to be divided into two ecdysteroid-dependent phases. Initially, moderate levels of ecdysteroid stimulate proliferation. Later, high levels of 20-hydroxyecdysone trigger a wave of apoptosis within the anlage that marks completion of its proliferative phase (Champlin, 1998).

The optic lobe of Manduca sexta, like that of Drosophila, is composed of three ganglia, the lamina, medulla and lobula. The neurons of the optic ganglia are progeny of neuroblasts in the optic lobe anlage (OA). In Manduca, the neuroblasts are arranged in a double-banded bracelet that comprises the inner and outer OA. During early larval stages, expansion of the neuroblast population occurs by symmetric cell divisions. In the final larval instar, the neuroblasts switch to the asymmetric divisions that lead to neuron production. The smaller daughter of each asymmetric division, the ganglion mother cell (GMC), divides to produce neurons. GMCs of the inner OA produce the neurons of the lobula. The medial margin of the outer OA is composed of the neuroblasts and GMCs that produce the neurons of the medulla (medulla precursor cells, MPC), while the lateral margin is composed of the neuroblasts and GMCs that produce the neurons of the lamina (lamina precursor cells, LPC) (Champlin, 1998).

Proliferation of the LPC has been shown to be regulated by incoming retinal afferents. Transection of the optic nerve in Manduca also leads to disruption of LPC proliferation. Communication via retinal afferents provides a coordinating link between the developing retina and production of lamina neurons. Ecdysteroids, by contrast, control production of neurons throughout the optic lobe. The precursors for the medullar and lobular neurons both require ecdysteroids for proliferation. The optic nerve is severed in cultures making it difficult to determine if the same is true for the LPCs. Despite these conditions, , arrested LPCs still enter mitosis in response to 20E. It appears that neural precursors throughout the optic lobe have a similar ecdysone-dependent G2 checkpoint. The failure of LPCs in cultured brains to incorporate BUdR suggests that 20E allows the cells to divide but they then arrest in G1 in the absence of innervation. This interpretation is consistent with data from Drosophila that LPCs arrest in G1 in mutants lacking proper retinal innervation. Thus, the cell cycle of LPCs in Manduca may have two developmentally regulated control points, stimulation by retinal afferents being required for progression through G1 and stimulation by ecdysteroids being required for progression through G2 (Champlin, 1998).

MPCs are found to proliferate in an ecdysteroid-dependent manner, even in small pieces of the outer OA, clearly indicating that other long-range signals are not required for these cells. Cells throughout the OA, including the MPCs, express the ecdysteroid receptor; therefore, these cells could be responding directly to ecdysteroids. An important issue is whether the ecdysteroid-dependent proliferation of the MPC is a direct response to the steroid or due to short-range signals from neighboring cells. Evidence for communication between surrounding glia and neuroblasts is seen for the protein product of the anachronism locus, which is secreted by nearby glial cells and inhibits proliferation of optic lobe neuroblasts in Drosophila. The fact that the MPCs appear to respond to ecdysteroid as a unit suggests that there may be coordinating signals within the OA of Manduca (Champlin, 1998).

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 medulla^KS74 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, shi^ts1. 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 Vam^KS74. 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/shi^ts1 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 shi^ts1 expression in T1 might block a presumed nonsynaptic output from T1. Expressing shi^ts1 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 shi^ts1 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 shi^ts1 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 shi^ts1 did not block T1 output. Expression of DTI and Kir^2.1 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 shibire^ts. 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 shibire^ts. 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 shibire^ts. 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 shibire^ts 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., 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).

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/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).

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 ort^C1-3-Gal4 and ort^C1-3-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 ort^C1-3-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 ort^C1-4-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 ort^C1-4-Gal4-driven Ort expression restored a preference for UV (Attr_UV/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 ort^C1-3-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. ort^C1-4-Gal4 or ort^C1-3-LexA::VP16 driving a temperature-sensitive allele of shibire, shi^ts1, 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 ort^C1-3-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 ort^C1-3-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 (vGlut^OK371) 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, vGlut^OK371-dVP16AD, in combination with a general neuronal hemidriver, elav-Gal4DBD, drove expression in a pattern essentially identical to that driven by vGlut^OK371-Gal4, indicating that the vGlut^OK371-dVP16AD enhancer trap recapitulated the expression pattern of the vGlut^OK371-Gal4 driver. The combination of the vGlut^OK371-dVP16AD and ort^C1-3-Gal4DBD hemidrivers (designated ort^C1-3∩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 ort^C1-3∩vGlut driver was expressed in Dm8, Tm5c, and L1 neurons, as well as in the medulla glia-like cells. In contrast, cha∩ort^C1-3, the combination of cha-Gal4DBD choline acetyltransferase-Gal4DBD) and ort^C1-3-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 ort^C1-3∩vGlut driver restored normal UV preference in ort mutants. In contrast, expressing Ort in cholinergic Ort-expressing neurons using the cha∩ort^C1-3 driver further reduced UV preference, suggesting that the cholinergic Ort-expressing neurons reduce UV attraction or, more likely, enhance green attraction. Although the cha∩ort^C1-3 and ort^C1-3∩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 shi^ts1 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 ort^C2-Gal4 drove expression in Dm8 and L1-L3 but not in any Tm neurons, while ort^C3-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 ort^C2 or ort^C3 neuron subsets were sufficient and/or required for UV preference was examined. It was found that expressing Ort using the ort^C2-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 shi^ts1. It was found that flies carrying ort^C2->shi^ts1 exhibited strongly attenuated UV preference at the non-permissive, but not permissive, temperatures, indicating that the ort^C2 subset is required for normal UV preference. In contrast, restoring the ort^C3 subset activity further reduced UV preference, suggesting that the ort^C3 subset inhibits UV sensing, or enhances green-sensing pathways. Moreover, blocking the activity of the ort^C3 subset using shi^ts1 did not confer a stronger UV preference, suggesting that the ort^C3 subset is sufficient but likely not required for phototactic preference to green light (Gao, 2009).

The preceding evidence indicated that the two lines, ort^C2 and ort∩vGlut, together identified the Dm8 neurons both functionally and anatomically as a substrate for UV preference. To test this possibility directly, an ort^C2-Gal4^DBD hemidriver was generated and combined with the vGlut-dVP16AD hemidriver. It was found that the combinatorial driver ort^C2∩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->shi^ts1 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 ort^C2-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∩ort^C1-3 or ort^C3-Gal4 driver significantly reduced UV preference in ort mutants, and Tm9 is covered by both drivers. Furthermore, inactivating Tm9 using the ort^C3 driver and shi^ts1 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 ort^C3 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).

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-BSH 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).

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