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.

orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control.

BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.

FENS-Kavli Network of Excellence: Science needs strong European cohesion.

Decades of scientific collaboration have brought innovation, prosperity and wide societal benefit to Europe. However, recent political events have impacted pan-European research and collaborations, and solutions are yet to materialise. Here, we argue that a vibrant, united European Research community led by its members and independent from political bodies is needed for Europe to remain a successful, interconnected scientific hub and keep delivering globally competitive science. The Federation of European Neuroscience Societies (FENS) is in an ideal position to play a paramount role in this endeavour.

Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria.

The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. Here, we exploit the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive behaviour. Contrary to expectations, we found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, we show that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection.

A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila.

Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. Here, we describe how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. We show that sex- and food-deprived male flies prioritize feeding over courtship initiation, and manipulation of food quality or the animal's internal state fine-tunes this decision. We identify the tyramine signaling pathway as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly's nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. We propose that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner.

Activation of Latent Courtship Circuitry in the Brain of Drosophila Females Induces Male-like Behaviors.

Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors [1, 2]. Fruit fly males exhibit an elaborate courtship display toward a potential mate [1, 2]. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru) [1]. Activation of male-specific dsx/fru(+) P1 neurons in the brain initiates the male's courtship display [3, 4], suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx(+) neurons play a pivotal role in sexual receptivity and post-mating behaviors [1, 2, 5-9]. Yet it is still unclear how dsx(+) neurons and dimorphisms in these circuits give rise to the different behaviors displayed by males and females. Here, we manipulated the function of dsx(+) neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, we found that activation of female dsx(+) neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. We uncovered specific dsx(+) neurons critical for driving male courtship and identified pheromones that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, our study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.

Sexually dimorphic octopaminergic neurons modulate female postmating behaviors in Drosophila.

Mating elicits profound behavioral and physiological changes in many species that are crucial for reproductive success. After copulation, Drosophila melanogaster females reduce their sexual receptivity and increase egg laying [1, 2]. Transfer of male sex peptide (SP) during copulation mediates these postmating responses [1, 3-6] via SP sensory neurons in the uterus defined by coexpression of the proprioceptive neuronal marker pickpocket (ppk) and the sex-determination genes doublesex (dsx) and fruitless (fru) [7-9]. Although neurons expressing dsx downstream of SP signaling have been shown to regulate postmating behaviors [9], how the female nervous system coordinates the change from pre- to postcopulatory states is unknown. Here, we show a role of the neuromodulator octopamine (OA) in the female postmating response. Lack of OA disrupts postmating responses in mated females, while increase of OA induces postmating responses in virgin females. Using a novel dsx(FLP) allele, we uncovered dsx neuronal elements associated with OA signaling involved in modulation of postmating responses. We identified a small subset of sexually dimorphic OA/dsx(+) neurons (approximately nine cells in females) in the abdominal ganglion. Our results are consistent with a model whereby OA neuronal signaling increases after copulation, which in turn modulates changes in female behavior and physiology in response to reproductive state.

Circadian period integrates network information through activation of the BMP signaling pathway.

Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

Neural circuitry underlying Drosophila female postmating behavioral responses.

BACKGROUND: After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. RESULTS: We demonstrate an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. We uncovered shared circuitry between dsx and a subset of the previously described SP-responsive fru(+)/ppk(+)-expressing neurons in the reproductive system. In addition, we identified sexually dimorphic dsx circuitry within the abdominal ganglion (Abg) critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. CONCLUSIONS: We propose that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs.

Invertebrate neuroethology: food play and sex.

How do animals perceive their environment and make appropriate behavioral choices based on those perceptions? New data have uncovered a novel sensory pathway that promotes Drosophila male courtship behavior in response to food.

ENA/VASP downregulation triggers cell death by impairing axonal maintenance in hippocampal neurons.

Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration.

A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.

Neuronal death in Drosophila triggered by GAL4 accumulation.

The GAL4/UAS system has been extensively employed in Drosophila to control gene expression in defined spatial patterns. More recently this system has been successfully applied to express genes involved in neurodegeneration to model various diseases in the fruit fly. We used transgenic lines expressing different levels of GAL4 in a particular subset of neurons involved in the control of rhythmic behaviour, so that its impact on neuronal physiology would result in altered locomotor activity, which could be readily assessed. We observed a striking correlation between gal4 dosage and behavioural defects associated with apoptotic neuronal loss in the specific GAL4-expressing neurons. Increased gal4 dosage correlated with accumulation of insoluble GAL4, suggesting that the cascade of events leading to apoptosis might be triggered by protein deposits of either GAL4 or protein intermediates. Behavioural defects were rescued by expression of hsp70, a classic chaperone that also interferes with cell death pathways. In agreement with the latter, the viral caspase inhibitor p35 also rescued GAL4-induced behavioural defects. Our observations demonstrate the intrinsic effects of GAL4 deregulation on neuronal viability and suggest that an excess of GAL4 might enhance neuronal deficits observed in models of neurodegeneration.

Delta-aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines.

BACKGROUND: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. RESULTS: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.