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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.

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.

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.

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.

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.