BACKGROUND: Detecting the direction of visual motion is an essential task of the early visual system. The Reichardt detector has been proven to be a faithful description of the underlying computation in insects. A series of recent studies addressed the neural implementation of the Reichardt detector in Drosophila revealing the overall layout in parallel ON and OFF channels, its input neurons from the lamina (L1→ON, and L2→OFF), and the respective output neurons to the lobula plate (ON→T4, and OFF→T5). While anatomical studies showed that T4 cells receive input from L1 via Mi1 and Tm3 cells, the neurons connecting L2 to T5 cells have not been identified so far. It is, however, known that L2 contacts, among others, two neurons, called Tm2 and L4, which show a pronounced directionality in their wiring. RESULTS: We characterized the visual response properties of both Tm2 and L4 neurons via Ca(2+) imaging. We found that Tm2 and L4 cells respond with an increase in activity to moving OFF edges in a direction-unselective manner. To investigate their participation in motion vision, we blocked their output while recording from downstream tangential cells in the lobula plate. Silencing of Tm2 and L4 completely abolishes the response to moving OFF edges. CONCLUSIONS: Our results demonstrate that both cell types are essential components of the Drosophila OFF motion vision pathway, prior to the computation of directionality in the dendrites of T5 cells.

BACKGROUND: Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms. RESULTS: We show that most fru(+) cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fru(C) mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on Fru(C) rather than other single Fru isoforms. Removal of Fru(C) from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark. CONCLUSIONS: These data map specific aspects of courtship behavior to the level of single fru isoforms and fru(+) cell types-an important step toward elucidating the chain of causality from gene to circuit to behavior.

The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. We circumvent this problem by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5 (ref. 2). Here we find that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours.

Different stimulus intensities elicit distinct perceptions, implying that input signals are either conveyed through an overlapping but distinct subpopulation of sensory neurons or channeled into divergent brain circuits according to intensity. In Drosophila, carbon dioxide (CO2) is detected by a single type of olfactory sensory neuron, but information is conveyed to higher brain centers through second-order projection neurons (PNs). Two distinct pathways, PN(v)-1 and PN(v)-2, are necessary and sufficient for avoidance responses to low and high CO2 concentrations, respectively. Whereas low concentrations activate PN(v)-1, high concentrations activate both PN(v)s and GABAergic PN(v)-3, which may inhibit PN(v)-1 pathway-mediated avoidance behavior. Channeling a sensory input into distinct neural pathways allows the perception of an odor to be further modulated by both stimulus intensity and context.

How the brain perceives sensory information and generates meaningful behavior depends critically on its underlying circuitry. The protocerebral bridge (PB) is a major part of the insect central complex (CX), a premotor center that may be analogous to the human basal ganglia. Here, by deconstructing hundreds of PB single neurons and reconstructing them into a common three-dimensional framework, we have constructed a comprehensive map of PB circuits with labeled polarity and predicted directions of information flow. Our analysis reveals a highly ordered information processing system that involves directed information flow among CX subunits through 194 distinct PB neuron types. Circuitry properties such as mirroring, convergence, divergence, tiling, reverberation, and parallel signal propagation were observed; their functional and evolutional significance is discussed. This layout of PB neuronal circuitry may provide guidelines for further investigations on transformation of sensory (e.g., visual) input into locomotor commands in fly brains.

Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist.

Learning through trial-and-error interactions allows animals to adapt innate behavioural ‘rules of thumb’ to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. Here we show that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and we identify a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. Our work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience.

Females of many animal species emit chemical signals that attract and arouse males for mating. For example, the major aphrodisiac pheromone of Drosophila melanogaster females, 7,11-heptacosadiene (7,11-HD), is a potent inducer of male-specific courtship and copulatory behaviors. Here, we demonstrate that a set of gustatory sensory neurons on the male foreleg, defined by expression of the ppk23 marker, respond to 7,11-HD. Activity of these neurons is required for males to robustly court females or to court males perfumed with 7,11-HD. Artificial activation of these ppk23(+) neurons stimulates male-male courtship even without 7,11-HD perfuming. These data identify the ppk23(+) sensory neurons as the primary targets for female sex pheromones in Drosophila.

The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain. How these axons select a specific pathway is not known. Here, we used a microarray-based approach to identify genes expressed in R8 neurons as they extend into the brain. We found that Roundabout-3 (Robo3), an axon-guidance receptor, is expressed specifically and transiently in R8 growth cones. In wild-type animals, posterior-most R8 axons extend along a border of glial cells demarcated by the expression of Slit, the secreted ligand of Robo3. In contrast, robo3 mutant R8 axons extend across this border and fasciculate inappropriately with other axon tracts. We demonstrate that either Robo1 or Robo2 rescues the robo3 mutant phenotype when each is knocked into the endogenous robo3 locus separately, indicating that R8 does not require a function unique to the Robo3 paralog. However, persistent expression of Robo3 in R8 disrupts the layer-specific targeting of R8 growth cones. Thus, the transient cell-specific expression of Robo3 plays a crucial role in establishing neural circuits in the Drosophila visual system by selectively regulating pathway choice for posterior-most R8 growth cones.

The courtship song of the Drosophila male serves as a genetically tractable model for the investigation of the neural mechanisms of decision-making, action selection, and motor pattern generation. Singing has been causally linked to the activity of the set of neurons that express the sex-specific fru transcripts, but the specific neurons involved have not been identified. Here we identify five distinct classes of fru neuron that trigger or compose the song. Our data suggest that P1 and pIP10 neurons in the brain mediate the decision to sing, and to act upon this decision, while the thoracic neurons dPR1, vPR6, and vMS11 are components of a central pattern generator that times and shapes the song’s pulses. These neurons are potentially connected in a functional circuit, with the descending pIP10 neuron linking the brain and thoracic song centers. Sexual dimorphisms in each of these neurons may explain why only males sing.