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Inhibitory neurons are essential for nervous system function. GABA is the most important neurotransmitter for fast neuronal inhibition in vertebrates and invertebrates. GABAergic signaling in sex specific,fruitlessexpressing neuronal circuits ofDrosophilais required for multiple aspects of male courtship behavior. RNAi mediated knockdown of the GABA producing enzyme Gad1 and the ionotropic receptor Rdl in the ventral nerve cord causes uncoordinated and futile copulation attempts, defects in wing extension choice and severe alterations of courtship song. Altered song of GABA depleted males fails to stimulate female receptivity, but rescue of song patterning alone is not sufficient to rescue male mating success. Knockdown of Gad1 and Rdl in brain circuits abolishes courtship conditioning. We characterize the around 220 neurons coexpressing Gad1 and Fruitless in theDrosophilamale nervous system and propose inhibitory circuit motifs underlying key features of courtship behavior based on the observed phenotypes.

Drosophila melanogaster produce different communication sounds during interactions with conspecifics by vibrating their wings. Males sing a pre-copulatory courtship song with pulse and sine elements. Females sing a structurally different song during copulation. Both sexes also produce a third type of sound pulses during agonistic interactions, i.e. during sexual rejection and inter-male aggression. It is well established that male courtship song increases female receptivity to mating. Female copulation song depends on male inseminate composition and might influence the transfer of the latter. The behavioural effect and function of agonistic sounds are not yet fully understood. Male courtship song production relies on sex specific neurons. As a precisely quantifiable and very specific element of courtship with defined motor parameters, it is an ideal model system for behavioural circuit neuroscience and has provided basic insight to how the nervous system generates, modulates and patterns adaptive behaviour. Central brain networks integrate multimodal sensory information and flexibly control the initiation and maintenance of singing. The circuits generating the species-specific song pattern and shaping motor control of the wing are mainly located in the ventral nerve cord. First studies have started to address how other Drosophila species with divergent sound producing behaviour differ on the level of neuronal circuits from D. melanogaster. In the future, advances in connectomics are expected to greatly facilitate neuronal circuit analysis in general and allow for a comprehensive synthesis of described circuit motifs for different aspects of sound production and its coordination in behavioural context.

Females communicate sexual receptivity in various ways. Drosophila signal that they are mated and ovulating, and resistive to mating again, by extruding their egg-laying organ (ovipositor). Connectome-aided circuit analysis reveals how this break up message is computed and differs from an acceptance response in virgins.

Foraging animals have to evaluate, compare and select food patches in order to increase their fitness. Understanding what drives foraging decisions requires careful manipulation of the value of alternative options while monitoring animals choices. Value-based decision-making tasks in combination with formal learning models have provided both an experimental and theoretical framework to study foraging decisions in lab settings. While these approaches were successfully used in the past to understand what drives choices in mammals, very little work has been done on fruit flies. This is despite the fact that fruit flies have served as model organism for many complex behavioural paradigms. To fill this gap we developed a single-animal, trial-based decision making task, where freely walking flies experienced optogenetic sugar-receptor neuron stimulation. We controlled the value of available options by manipulating the probabilities of optogenetic stimulation. We show that flies integrate reward history of chosen options and forget value of unchosen options. We further discover that flies assign higher values to rewards experienced early in the behavioural session, consistent with formal reinforcement learning models. Finally, we also show that the probabilistic rewards affect walking trajectories of flies, suggesting that accumulated value is controlling the navigation vector of flies in a graded fashion. These findings establish the fruit fly as a model organism to explore the genetic and circuit basis of reward foraging decisions.

In most animal species, males and females communicate during sexual behavior to negotiate reproductive investments. Pre-copulatory courtship may settle if copulation takes place, but often information exchange and decision-making continue beyond that point. Here, we show that female Drosophila sing by wing vibration in copula. This copulation song is distinct from male courtship song and requires neurons expressing the female sex determination factor DoublesexF. Copulation song depends on transfer of seminal fluid components of the male accessory gland. Hearing female copulation song increases the reproductive success of a male when he is challenged by competition, suggesting that auditory cues from the female modulate male ejaculate allocation. Our findings reveal an unexpected fine-tuning of reproductive decisions during a multimodal copulatory dialog. The discovery of a female-specific acoustic behavior sheds new light on Drosophila mating, sexual dimorphisms of neuronal circuits and the impact of seminal fluid molecules on nervous system and behavior., Male fruitflies sing a patterned wing song during courtship. Here, the authors show that females sing a distinct song during copulation, which is controlled by sex-specific neurons, depends on seminal fluid from the male accessory gland and modulates latency of female remating with subsequent males.

Animals often navigate environments that are uncertain, volatile and complex, making it challenging to locate reliable food sources. Therefore, it is not surprising that many species evolved multiple, parallel and complementary foraging strategies to survive. Current research on animal behavior is largely driven by a reductionist approach and attempts to study one particular aspect of behavior in isolation. This is justified by the huge success of past and current research in understanding neural circuit mechanisms of behaviors. But focusing on only one aspect of behaviors obscures their inherent multidimensional nature. To fill this gap we aimed to identify and characterize distinct behavioral modules using a simple reward foraging assay. For this we developed a single-animal, trial-based probabilistic foraging task, where freely walking fruit flies experience optogenetic sugar-receptor neuron stimulation. By carefully analyzing the walking trajectories of flies, we were able to dissect the animals foraging decisions into multiple underlying systems. We show that flies perform local searches, cue-based navigation and learn task relevant contingencies. Using probabilistic reward delivery allowed us to bid several competing reinforcement learning (RL) models against each other. We discover that flies accumulate chosen option values, forget unchosen option values and seek novelty. We further show that distinct behavioral modules -learning and navigation-based systems-cooperate, suggesting that reinforcement learning in flies operates on dimensionality reduced representations. We therefore argue that animals will apply combinations of multiple behavioral strategies to generate foraging decisions.

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.

Behavioral neurobiology aims at explaining behavior at the level of neurons and neuronal circuits, based on linking comparative anatomy, and the observation and manipulation of nervous system activity with animal behavior. The numerical simplicity and the presence of identified neurons in insect nervous systems make them outstanding model systems for neurobiology. The insect nervous system has a common ground plan with functionally specialized regions for sensory processing, integration, and motor control. In holometabolous species, the nervous system is restructured during metamorphosis to support new behavior. Different forms of plasticity allow for behavioral adaptations in the adult stage. Neuronal circuits for behavior in Drosophila melanogaster can be effectively analysed with genetic tools, as exemplified for courtship and mating behavior. Recent developments in connectomics and genome editing are expected to further behavioral neurobiology in a broad range of species and permit a comprehensive comparative approach to the neurobiology of behavioral ecology.

Multifunctional motor systems produce distinct output patterns that are dependent on behavioral context, posing a challenge to underlying neuronal control. Flies use their wings for flight and the production of a patterned acoustic signal, the male courtship song, employing in both cases a small set of wing muscles and corresponding motor neurons. We took first steps toward elucidating the neuronal control mechanisms of this multifunctional motor system by live imaging of muscle ensemble activity patterns during song and flight, and we established the functional role of a comprehensive set of wing muscle motor neurons by silencing experiments. Song and flight rely on distinct configurations of neuromuscular activity, with most, but not all, flight muscles and their corresponding motor neurons contributing to song and shaping its acoustic parameters. The two behaviors are exclusive, and the neuronal command for flight overrides the command for song. The neuromodulator octopamine is a candidate for selectively stabilizing flight, but not song motor patterns.

Drosophila melanogaster sexual behaviour relies on well-studied genetically determined neuronal circuits. At the same time, it can be flexible and is modulated by multiple external and internal factors. This review focuses on how physiological state, behavioural context and social experience impact sexual circuits in the two sexes. We discuss how females tune receptivity and other behaviours depending on mating status and how males adjust courtship intensity based on sexual satiety, age and the conflicting drive for aggression. Neuronal mechanisms for behavioural modulation include changes in sensory and central processing. Activity of modulatory neurons can enhance, suppress or reverse the behavioural response to sensory cues. In summary, fly sexual behaviour is an excellent model to study mechanisms of neuromodulation of complex innate behaviour on the circuit level.

Genetic hardwiring during brain development provides computational architectures for innate neuronal processing. Thus, the paradigmatic chick retinotectal projection, due to its neighborhood preserving, topographic organization, establishes millions of parallel channels for incremental visual field analysis. Retinal axons receive targeting information from quantitative guidance cue gradients. Surprisingly, novel adaptation assays demonstrate that retinal growth cones robustly adapt towards ephrin-A/EphA forward and reverse signals, which provide the major mapping cues. Computational modeling suggests that topographic accuracy and adaptability, though seemingly incompatible, could be reconciled by a novel mechanism of coupled adaptation of signaling channels. Experimentally, we find such ‘co-adaptation’ in retinal growth cones specifically for ephrin-A/EphA signaling. Co-adaptation involves trafficking of unliganded sensors between the surface membrane and recycling endosomes, and is presumably triggered by changes in the lipid composition of membrane microdomains. We propose that co-adaptative desensitization eventually relies on guidance sensor translocation into cis-signaling endosomes to outbalance repulsive trans-signaling. DOI: http://dx.doi.org/10.7554/eLife.25533.001, eLife digest The human brain contains roughly 100 billion neurons, which are organized into complex networks. But how does the brain establish these networks in the first place? Neurons have long projections known as axons and, in the developing brain, these axons form structures called growth cones at their tips. The growth cones possess finger-like appendages that probe their surroundings in search of signals displayed on the surface of other cells. These signals guide the growth cones to their targets and move the axon tip into a position where it can form connections with other neurons within a particular network. The signals that growth cones follow are often distributed in concentration gradients so that the levels of a signal may be low at one end of a brain structure and gradually increase to a maximum level at the other end. In the developing visual system, for example, about one million axons from the retina reach their proper targets in visual regions of the brain by reading gradients of signals called ephrins and Ephs. However, when Fiederling et al. studied retinal neurons in a petri dish, they found that the axons became much less sensitive to both signals upon prolonged exposure to them. This unexpected finding raised a new question. If neurons rely upon these gradients for navigation, how do they continue to find their way if they also become less sensitive to those signals over time? Fiederling et al. used a computer to simulate the events occurring in the developing brain. The simulations were based on the idea that navigating growth cones sense the ratio of ephrins to Ephs, instead of sensing the individual concentrations of these signals. Thus, by keeping the amounts of all involved sensors in strict proportion to each other while continuously re-adjusting them, the axons could still be accurately guided to their targets even though the neurons would become less sensitive to the signals. Experiments in neurons grown in petri dishes confirmed that retinal growth cones do exactly this and regulate the amounts of ephrin and Eph sensors on their outer membranes in a highly coordinated manner using a previously unknown mechanism. Given that signaling requires energy, the brain may have evolved this system to reduce the costs associated with wiring itself up. The system also offers greater flexibility than guidance based on the absolute concentrations of the signals. If other regions of the brain use a similar mechanism to establish their own wiring patterns, then understanding such basic mechanisms might eventually provide insights into diseases of miswiring such as schizophrenia and autism. DOI: http://dx.doi.org/10.7554/eLife.25533.002

SummaryThe 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.

A microfluidic network (microFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (microCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 microm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5.

Although males are generally less discriminating than females when it comes to choosing a mate, they still benefit from distinguishing between mates that are receptive to courtship and those that are not, in order to avoid wasting time and energy. It is known that males of Drosophila melanogaster are able to learn to associate olfactory and gustatory cues with female receptivity, but the role of more arbitrary, visual cues in mate choice learning has been overlooked to date in this species. We therefore carried out a series of experiments to determine: 1) whether males had a baseline preference for female eye color (red versus brown), 2) if males could learn to associate an eye color cue with female receptivity, and 3) whether this association disappeared when the males were unable to use this visual cue in the dark. We found that naïve males had no baseline preference for females of either eye color, but that males which were trained with sexually receptive females of a given eye color showed a preference for that color during a standard binary choice experiment. The learned cue was indeed likely to be truly visual, since the preference disappeared when the binary choice phase of the experiment was carried out in darkness.This is, to our knowledge 1) the first evidence that male D. melanogaster can use more arbitrary cues and 2) the first evidence that males use visual cues during mate choice learning. Our findings suggest that that D. melanogaster has untapped potential as a model system for mate choice learning.

Microcontact printing (microCP) of proteins has been successfully used for patterning surfaces in various contexts. Here we describe a simple 'lift-off' method to print precise patterns of axon guidance molecules, which are used as substrate for growing chick retinal ganglion cell (RGC) axons. Briefly, the etched pattern of a silicon master is transferred to a protein-coated silicone cuboid (made from polydimethylsiloxane, PDMS), which is then used as a stamp on a glass coverslip. RGC explants are placed adjacent to the pattern and cultured overnight. Fluorescent labeling of the printed proteins allows the quantitative analysis of the interaction of axons and growth cones with single protein dots and of the overall outgrowth and guidance rate in variously designed patterns. Patterned substrates can be produced in 3-4 h and are stable for up to one week at 4 degrees C; the entire protocol can be completed in 3 d.

Summary 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 fruC 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 FruC rather than other single Fru isoforms. Removal of FruC 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., Highlights • fru A, B, and C isoforms have largely overlapping expression in the male fly CNS • All three fru isoforms contribute to male courtship, with fruC being the most critical • FruC specifies sexual dimorphisms in neuron number and arborizations • FruC is required in defined neuronal classes for male-specific anatomy and behavior, Drosophila express three male-specific isoforms (A, B, and C) of the putative transcription factor fruitless (fru) in an overlapping pattern. von Philipsborn et al. show that isoform-mutant flies exhibit defects in male courtship, copulation success, and song production. FruC specifies sexual dimorphisms in defined neuronal classes potentially crucial for pheromone processing.

International audience; Slit proteins are secreted ligands that interact with the Roundabout (Robo) receptors to provide important guidance cues in neuronal and vascular development. Slit-Robo signalling is mediated by an interaction between the second Slit domain and the first Robo domain, as well as being dependent on heparan sulphate. In an effort to understand the role of the other Slit domains in signalling, we determined the crystal structure of the fourth Slit2 domain (D4) and examined the effects of various Slit2 constructs on chick retinal ganglion cell axons. Slit2 D4 forms a homodimer using the conserved residues on its concave face, and can also bind to heparan sulphate. We observed that Slit2 D4 frequently results in growth cones with collapsed lamellipodia and that this effect can be inhibited by exogenously added heparan sulphate. Our results show that Slit2 D4-heparan sulphate binding contributes to a Slit-Robo signalling mechanism more intricate than previously thought.

Graded distributions of proteins are pivotal for many signaling processes during development, such as morphogenesis, cell migration, and axon guidance. Here, we describe a technique to fabricate substrate-bound stepwise protein gradients by means of a microfluidic network etched into a silicon wafer with an array of parallel 14-micrometer–wide channels, which can be filled with a series of arbitrarily chosen protein solutions. In a subsequent microcontact printing step, the protein pattern is transferred onto a surface and is used as a substrate for cell culture. Cellular responses to a defined microscopic pattern of a protein, such as guided axonal outgrowth and directed migration, cell polarization, changes in morphology, and signaling, can be thus studied in a controlled in vitro environment.

Although males are generally less discriminating than females when it comes to choosing a mate, they still benefit from distinguishing between mates that are receptive to courtship and those that are not, in order to avoid wasting time and energy. It is known that males of Drosophila melanogaster are able to learn to associate olfactory and gustatory cues with female receptivity, but the role of more arbitrary, visual cues in mate choice learning has been overlooked to date in this species. We therefore carried out a series of experiments to determine: 1) whether males had a baseline preference for female eye color (red versus brown), 2) if males could learn to associate an eye color cue with female receptivity, and 3) whether this association disappeared when the males were unable to use this visual cue in the dark. We found that naïve males had no baseline preference for females of either eye color, but that males which were trained with sexually receptive females of a given eye color showed a preference for that color during a standard binary choice experiment. The learned cue was indeed likely to be truly visual, since the preference disappeared when the binary choice phase of the experiment was carried out in darkness.This is, to our knowledge 1) the first evidence that male D. melanogaster can use more arbitrary cues and 2) the first evidence that males use visual cues during mate choice learning. Our findings suggest that that D. melanogaster has untapped potential as a model system for mate choice learning.