GABA transmission from mAL interneurons regulates aggression in Drosophila males.

Aggression is known to be regulated by pheromonal information in many species. But how central brain neurons processing this information modulate aggression is poorly understood. Using the fruit fly model of Drosophila melanogaster, we systematically characterize the role of a group of sexually dimorphic GABAergic central brain neurons, popularly known as mAL, in aggression regulation. The mAL neurons are known to be activated by male and female pheromones. In this report, we show that mAL activation robustly increases aggression, whereas its inactivation decreases aggression and increases intermale courtship, a behavior considered reciprocal to aggression. GABA neurotransmission from mAL is crucial for this behavior regulation. Exploiting the genetic toolkit of the fruit fly model, we also find a small group of approximately three to five GABA+ central brain neurons with anatomical similarities to mAL. Activation of the mAL resembling group of neurons is necessary for increasing intermale aggression. Overall, our findings demonstrate how changes in activity of GABA+ central brain neurons processing pheromonal information, such as mAL in Drosophila melanogaster, directly modulate the social behavior of aggression in male-male pairings.

A small number of cholinergic neurons mediate hyperaggression in female Drosophila.

In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. We quantified the enhancement by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, we used an intersectional approach with our library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsxFLP instead of 357FLP for the intersectional studies, we found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression.

Masculinized Drosophila females adapt their fighting strategies to their opponent.

Many animal species show aggression to gain mating partners and to protect territories and other resources from competitors. Both male and female fruit flies of the species Drosophila melanogaster exhibit aggression in same-sex pairings, but the strategies used are sexually dimorphic. We have begun to explore the biological basis for the differing aggression strategies, and the cues promoting one form of aggression over the other. Here, we describe a line of genetically masculinized females that switch between male and female aggression patterns based on the sexual identity of their opponents. When these masculinized females are paired with more aggressive opponents, they increase the amount of male-like aggression they use, but do not alter the level of female aggression. This suggests that male aggression may be more highly responsive to behavioral cues than female aggression. Although the masculinized females of this line show opponent-dependent changes in aggression and courtship behavior, locomotor activity and sleep are unaffected. Thus, the driver line used may specifically masculinize neurons involved in social behavior. A discussion of possible different roles of male and female aggression in fruit flies is included here. These results can serve as precursors to future experiments aimed at elucidating the circuitry and triggering cues underlying sexually dimorphic aggressive behavior.

Putative transmembrane transporter modulates higher-level aggression in Drosophila.

By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

Single dopaminergic neurons that modulate aggression in Drosophila.

Monoamines, including dopamine (DA), have been linked to aggression in various species. However, the precise role or roles served by the amine in aggression have been difficult to define because dopaminergic systems influence many behaviors, and all can be altered by changing the function of dopaminergic neurons. In the fruit fly, with the powerful genetic tools available, small subsets of brain cells can be reliably manipulated, offering enormous advantages for exploration of how and where amine neurons fit into the circuits involved with aggression. By combining the GAL4/upstream activating sequence (UAS) binary system with the Flippase (FLP) recombination technique, we were able to restrict the numbers of targeted DA neurons down to a single-cell level. To explore the function of these individual dopaminergic neurons, we inactivated them with the tetanus toxin light chain, a genetically encoded inhibitor of neurotransmitter release, or activated them with dTrpA1, a temperature-sensitive cation channel. We found two sets of dopaminergic neurons that modulate aggression, one from the T1 cluster and another from the PPM3 cluster. Both activation and inactivation of these neurons resulted in an increase in aggression. We demonstrate that the presynaptic terminals of the identified T1 and PPM3 dopaminergic neurons project to different parts of the central complex, overlapping with the receptor fields of DD2R and DopR DA receptor subtypes, respectively. These data suggest that the two types of dopaminergic neurons may influence aggression through interactions in the central complex region of the brain involving two different DA receptor subtypes.

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila.

Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.

Sexually dimorphic peripheral sensory neurons regulate copulation duration and persistence in male Drosophila.

Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The peripheral sensory information required to regulate copulation duration is unclear. Here, we employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. We confirmed that these MSNs are cholinergic and co-express both fru and dsx. We found that the sensory information received by the peripheral sensory neurons from the front legs (GRNs) and mechanosensory neurons (MSNs) at the male genitalia contribute to the regulation of copulation duration. Moreover, our results show that their function is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete.

Specific subgroups of FruM neurons control sexually dimorphic patterns of aggression in Drosophila melanogaster.

A great challenge facing neuroscience is to understand how genes, molecules, cells, circuits, and systems interact to generate social behavior. Fruit flies (Drosophila melanogaster) offer a powerful model system to address questions of this magnitude. These animals display genetically specified, sexually dimorphic patterns of fighting behavior via sex-specific splicing of the fruitless gene. Here, we show that sexually dimorphic behavioral patterns displayed during aggression are controlled by specific subgroups of neurons expressing male forms of fruitless proteins (Fru(M)). Using the GAL4/UAS system to manipulate transformer expression, we feminized or masculinized different populations of neurons in fly nervous systems. With a panneuronal elav-GAL4 driver, male patterns of fighting behavior were transferred into females and female patterns into males. We screened 60 Gal4 lines that express the yeast transcription factor in different patterns in fly central nervous systems and found five that showed abnormal same-sex courtship behavior. The sexually dimorphic fighting patterns, however, were completely switched only in one and partially switched in a second of these lines. In the other three lines, female patterns of aggression were seen despite a switch in courtship preference. A tight correspondence was seen between Fru(M) expression and how flies fight in several subgroups of neurons usually expressing these proteins: Expression is absent when flies fight like females and present when flies fight like males, thereby beginning a separation between courtship and aggression among these neurons.

Serotonergic Modulation of Aggression in Drosophila Involves GABAergic and Cholinergic Opposing Pathways.

Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. Here, we identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. Our data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression.

Characterization of the Sexually Dimorphic fruitless Neurons That Regulate Copulation Duration.

Male courtship in Drosophila melanogaster is a sexually dimorphic innate behavior that is hardwired in the nervous system. Understanding the neural mechanism of courtship behavior requires the anatomical and functional characterization of all the neurons involved. Courtship involves a series of distinctive behavioral patterns, culminating in the final copulation step, where sperms from the male are transferred to the female. The duration of this process is tightly controlled by multiple genes. The fruitless (fru) gene is one of the factors that regulate the duration of copulation. Using several intersectional genetic combinations to restrict the labeling of GAL4 lines, we found that a subset of a serotonergic cluster of fru neurons co-express the dopamine-synthesizing enzyme, tyrosine hydroxylase, and provide behavioral and immunological evidence that these neurons are involved in the regulation of copulation duration.

Optogenetic Control of Gene Expression in Drosophila.

To study the molecular mechanism of complex biological systems, it is important to be able to artificially manipulate gene expression in desired target sites with high precision. Based on the light dependent binding of cryptochrome 2 and a cryptochrome interacting bHLH protein, we developed a split lexA transcriptional activation system for use in Drosophila that allows regulation of gene expression in vivo using blue light or two-photon excitation. We show that this system offers high spatiotemporal resolution by inducing gene expression in tissues at various developmental stages. In combination with two-photon excitation, gene expression can be manipulated at precise sites in embryos, potentially offering an important tool with which to examine developmental processes.

Single serotonergic neurons that modulate aggression in Drosophila.

Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, we restricted the population of 5HT neurons that can be reproducibly manipulated to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. Here, we identified a symmetrical pair of serotonergic PLP neurons that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggest that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. Our studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons.

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.

Feminizing cholinergic neurons in a male Drosophila nervous system enhances aggression.

Previous studies in Drosophila have demonstrated that whether flies fight like males or females can be switched by selectively manipulating genes of the sex determination hierarchy in male and female nervous systems. Here we extend these studies by demonstrating that changing the sex of cholinergic neurons in male fruit fly nervous systems via expression of the transformer gene increases the levels of aggression shown by the flies without altering the way the flies fight. Transformer manipulation in this way does not change phototaxis, geotaxis, locomotion or odor avoidance of the mutant males compared to controls. Cholinergic neurons must be feminized via this route during the late larval/early pupal stages of development to show the enhanced aggression phenotype. Other investigators have shown that this is the same time period during which sexually dimorphic patterns of behavior are specified in flies. Neurons that co-express fruitless and choline acetyl transferase are found in varying numbers within different clusters of fruitless-expressing neurons: together they make up approximately 10% of the pool of fruitless-expressing neurons in the brain and nerve cord.

Gender-selective patterns of aggressive behavior in Drosophila melanogaster.

Complex behaviors, such as aggression, are comprised of distinct stereospecific behavioral patterns (modules). How such patterns get wired into nervous systems remains unknown. Recently, we reported on a quantitative analysis of fighting behavior in male flies of the common Canton-S strain of Drosophila melanogaster. Here, we report a similar analysis of fighting behavior in females of the same species. Fights were carried out between pairs of virgin and pairs of mated females in competition for a yeast resource. Each fight was videotaped and analyzed by using transition matrices and Markov chain analyses. We observe only small difference in fighting intensity between virgin and mated females. In contrast to what is seen in male fights, however, no clear hierarchical relationship is formed in the female fights. A further comparison of the behavioral patterns making up male and female fights reveals that some modules are shared by both sexes, whereas others are highly selective. Within the shared components, transitions between the modules also show gender-selective differences. By using the powerful genetic methods available for examining behavior in fruit flies, it should be possible to use the gender-selective differences in fighting behavior to address the question of how these behavioral patterns get established in the brains of fruit flies.

Neuromuscular defects in a Drosophila survival motor neuron gene mutant.

Autosomal recessive spinal muscular atrophy (SMA) is linked to mutations in the survival motor neuron (SMN) gene. The SMN protein has been implicated at several levels of mRNA biogenesis and is expressed ubiquitously. Studies in various model organisms have shown that the loss of function of the SMN gene leads to embryonic lethality. The human contains two genes encoding for SMN protein and in patients one of these is disrupted. It is thought the remaining low levels of protein produced by the second SMN gene do not suffice and result in the observed specific loss of lower motor neurons and muscle wasting. The early lethality in the animal mutants has made it difficult to understand why primarily these tissues are affected. We have isolated a Drosophila smn mutant. The fly alleles contain point mutations in smn similar to those found in SMA patients. We find that zygotic smn mutant animals show abnormal motor behavior and that smn gene activity is required in both neurons and muscle to alleviate this phenotype. Physiological experiments on the fly smn mutants show that excitatory post-synaptic currents are reduced while synaptic motor neuron boutons are disorganized, indicating defects at the neuromuscular junction. Clustering of a neurotransmitter receptor subunit in the muscle at the neuromuscular junction is severely reduced. This new Drosophila model for SMA thus proposes a functional role for SMN at the neuromuscular junction in the generation of neuromuscular defects.