GABAergic signaling shapes multiple aspects of Drosophila courtship motor behavior.

Inhibitory neurons are essential for orchestrating and structuring behavior. We use one of the best studied behaviors in Drosophila, male courtship, to analyze how inhibitory, GABAergic neurons shape the different steps of this multifaceted motor sequence. RNAi-mediated knockdown of the GABA-producing enzyme GAD1 and the ionotropic receptor Rdl in sex specific, fruitless expressing neurons 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 male brain circuits abolishes courtship conditioning. We characterize the around 220 neurons coexpressing GAD1 and Fruitless in the Drosophila male nervous system and propose inhibitory circuit motifs underlying key features of courtship behavior based on the observed phenotypes.

Single-Pair Courtship and Competition Assays in Drosophila.

Courtship in Drosophila melanogaster involves a series of innate, complex behaviors that allow male and female flies to exchange sensory information and assess the quality of a potential mate. Although highly robust and stereotypical, courtship behaviors can be modulated by internal state and experience. This protocol describes methods for designing and carrying out experiments that measure courtship performance in single-pair assays, in which a male is paired with a female, or in competitive assays, in which a male is presented with a female and with a male competitor. It also includes approaches for measuring female sexual receptivity during courtship.

Courtship Conditioning/Suppression Assays in Drosophila.

Naive males court both virgin and mated females but learn through experience to discriminate between them, thus minimizing futile investments in nonreceptive female flies. In the laboratory, we can exploit the innate courtship enthusiasm of males and manipulate their behavior by placing them with a nonreceptive female (immature virgin females, decapitated mature virgin females, or mature mated females), termed as the courtship suppression/conditioning assay. Early studies showed that male flies that experience failure to mate upon interaction with nonreceptive previously mated females show decreased motivation to court (courtship suppression). Courtship suppression is an important experimental paradigm for studying genes and neuronal circuits that mediate short- and long-term memory. The anti-aphrodisiac male-specific pheromone 11-cis-vaccenyl-acetate plays a key role in this conditioned response, as male flies learn to associate its presence on mated females with the failure to mate.

Measurement of Drosophila Reproductive Behaviors.

Courtship behaviors in Drosophila melanogaster are innate and contain highly stereotyped but also experience- and state-dependent elements. They have been the subject of intense study for more than 100 years. The power of Drosophila as a genetic experimental system has allowed the dissection of reproductive behaviors at a molecular, cellular, and physiological level. As a result, we know a great deal about how flies perceive sensory cues from potential mates, how this information is integrated in higher brain centers to execute reproductive decisions, and how state and social contexts modulate these responses. The simplicity of the assay has allowed for its broad application. Here, we introduce methods for studying male and female innate reproductive behaviors as well as their plastic responses.

Female Fly Postmating Behaviors.

Upon copulation, females undergo a switch-like change in their behavior and physiology, known as "postmating responses." These strong behavioral and physiological changes are triggered by the transfer of male seminal proteins during copulation. Postmating response is associated with strong reduction in receptivity, indicated by the females kicking their legs toward the suitor and curving their abdomen downward to hide their genitalia from them and extruding their ovipositor at the tip of the abdomen, which physically prevents copulation. The transfer of male-specific pheromones, such as 11-cis-vaccenyl-acetate, during copulation further reduces female attractiveness. In addition, mated females exhibit increased ovulation, egg-laying behavior, enhanced feeding behavior, and changes in food preference. However, females increase their rate of remating when they are in social groups or in the presence of food. This protocol describes methods for measuring female postmating behaviors, such as oviposition, female sexual receptivity, and mating plug ejection.

Probing Acoustic Communication during Fly Reproductive Behaviors.

During reproduction, male and female flies use wing vibration to generate different acoustic signals. Males produce a courtship song before copulation that is easily recognized by unilateral wing vibration. In copula, females produce a distinct sound pattern (copulation song) with both wings. Sexual rejection of immature virgins and aggressive encounters between males are also accompanied by sound pulses generated by wing flicks. Fly song has frequency ranges audible to the human ear and can be directly listened to after appropriate amplification. When displayed in an oscillogram, audio recordings can be mapped on wing-movement patterns and thus provide a fast and precise method to sample and quantify motor behaviors with high temporal resolution. After recording different fly sounds, their effect on behavior can be tested in playback experiments.

Neuroscience: The Female Art of Saying No.

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.

Reward foraging task and model-based analysis reveal how fruit flies learn value of available options.

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.

Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

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.

Female copulation song is modulated by seminal fluid.

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.

Multifunctional Wing Motor Control of Song and Flight.

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.

Neuronal modulation of D. melanogaster sexual behaviour.

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.

Ephrin-A/EphA specific co-adaptation as a novel mechanism in topographic axon guidance.

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.

Male Drosophila melanogaster learn to prefer an arbitrary trait associated with female mating status.

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.

Cellular and behavioral functions of fruitless isoforms in Drosophila courtship.

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.

Neuronal control of Drosophila courtship song.

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.

Structure and functional relevance of the Slit2 homodimerization domain.

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.

Growth cone response to ephrin gradients produced by microfluidic networks.

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.

Substrate-bound protein gradients for cell culture fabricated by microfluidic networks and microcontact printing.

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.