Person:

Kravitz, Edward

Situations requiring rapid decision-making in response to dynamic environmental demands occur repeatedly in natural environments. Neuromodulation can offer important flexibility to the output of neural networks in coping with changing conditions, but the contribution of individual neuromodulatory neurons in social behavior networks remains relatively unknown. Here we manipulate the Drosophila octopaminergic system and assay changes in adult male decision-making in courtship and aggression paradigms. When the functional state of OA neural circuits is enhanced, males exhibit elevated courtship behavior towards other males in both behavioral contexts. Eliminating the expression of the male form of the neural sex determination factor, Fruitless (FruM), in three OA suboesophageal ganglia (SOG) neurons also leads to increased male-male courtship behavior in these same contexts. We analyzed the fine anatomical structure through confocal examination of labeled single neurons to determine the arborization patterns of each of the three FruM-positive OA SOG neurons. These neurons send processes that display mirror symmetric, widely distributed arbors of endings within brain regions including the ventrolateral protocerebra, the SOG and the peri-esophageal complex. The results suggest that a small subset of OA neurons have the potential to provide male selective modulation of behavior at a single neuron level.

Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of 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.

Chemosensory pheromonal information regulates aggression and reproduction in many species, but how pheromonal signals are transduced to reliably produce behavior is not well understood. Here we demonstrate that the pheromonal signals detected by Gr32a-expressing chemosensory neurons to enhance male aggression are filtered through octopamine (OA, invertebrate equivalent of norepinephrine) neurons. Using behavioral assays, we find males lacking both octopamine and Gr32a gustatory receptors exhibit parallel delays in the onset of aggression and reductions in aggression. Physiological and anatomical experiments identify Gr32a to octopamine neuron synaptic and functional connections in the suboesophageal ganglion. Refining the Gr32a-expressing population indicates that mouth Gr32a neurons promote male aggression and form synaptic contacts with OA neurons. By restricting the monoamine neuron target population, we show that three previously identified OA-FruM neurons involved in behavioral choice are among the Gr32a-OA connections. Our findings demonstrate that octopaminergic neuromodulatory neurons function as early as a second-order step in this chemosensory-driven male social behavior pathway.

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.