As in humans, the actions and reactions of male and female fruitflies during courtship are quite distinct. The differences seem to lie in gender-specific neural interpretations of the same sensory signals. See Letter p.686
The self-help relationship guide Men are from Mars, Women are from Venus1 stresses the differences in how men and women convey and interpret feelings through words and actions. During courtship, male and female Drosophila fruitflies also communicate distinct messages using several sensory stimuli (visual, chemical and acoustic) to evoke very different behaviours2. Three papers3,4,5, including one by Ruta et al.3 on page 686 of this issue, probe deeply into the neural circuits underlying fruitfly courtship and find that these gender-specific responses may be due not to differences in how external signals are initially sensed, but rather — perhaps similarly to ourselves — in how they are interpreted by the brain.
The mating ground for fruitflies is reminiscent of a Roman orgy: typically a crowded, overripe fruit where these animals feed. Males take the lead in deciding who to court, and assess other flies on the basis of their appearance, smell and taste2. On identifying a suitable partner — female, young (preferably virgin) and of the same species — the suitor serenades her with a love song produced by the vibration of one of his wings6. The behaviour of the female seems more passive, or at least too subtle for human voyeurs to detect easily; but it is she who ultimately decides whether to allow the male to initiate copulation.
These gender-specific differences in courtship behaviour are largely determined by a male-specific transcription factor called FruM, which is expressed in 1–2% of the more than 100,000 neurons in the fly nervous system7. Neurons expressing this factor (FruM neurons) include sensory cells for odours, tastes, sounds and sights; central neurons in the brain; and motor neurons that control wing and leg movements7,8. This hints that FruM neurons form an interconnected circuit7,8. Although FruM is expressed and essential only in males, female flies contain similar classes of neurons that are required for reproductive behaviours such as egg-laying9.
Surprisingly, initial work7 reported no dramatic sexual dimorphisms of these neural pathways — apart from differences in FruM expression — that could account for the distinct male and female courtship behaviours. More recent investigations10,11,12, however, found that small subpopulations of these neurons have gender-specific properties. The three new studies3,4,5 take a closer look at FruM neurons in males and their counterparts in females to address two fundamental questions: do they really form an interconnected circuit; and how widespread are sexual dimorphisms? To scrutinize these neural pathways, each team used a distinct approach — a worthy reminder of the power of fly genetics for dissecting brain structure and function.
Yu et al.4 devised an 'intersectional' genetic strategy to express reporter proteins in small, consistent subsets of FruM neurons in individual brains, which allowed them to visualize the projections of these neurons with greater clarity than before. Cachero et al.5 used a clonal marking method, in which subpopulations of FruM neurons that derive from the same neural stem cell were labelled.
The authors4,5 applied each approach exhaustively, to identify more than 100 distinct groups of FruM neurons throughout the nervous system. Moreover, they reconstructed in silico a comprehensive (although hypothetical) 'wiring diagram' of the FruM neural circuits, by digitally reconstructing the morphology of these neurons' projections — dendrites and axons, which receive and transmit neural signals, respectively — and by integrating the mapped neurons into a common reference brain (Fig. 1). This allowed them to predict the flow of information from one set of neurons to another, as well as to locate brain regions essential for integrating the diverse types of sensory message that pass between males and females during courtship.
In contrast to these 'global' analyses, Ruta et al.3 focused on a single olfactory pathway that is responsive to cis-vaccenyl acetate (cVA) — a male-specific pheromone that promotes sexual receptivity in females but inhibits courtship in males13. To visualize the neural components of this pathway, the authors expressed in all FruM neurons a photoactivatable reporter protein, PA–GFP, that can be converted from a low to a high fluorescence state with a pulse of high-energy light12. They then activated PA–GFP in precisely the brain area that is innervated by the axons of cVA-responsive olfactory sensory neurons. As this region also contains the dendrites of neurons to which these sensory cells connect, PA–GFP was simultaneously activated in this second population of neurons, and its diffusion revealed the neurons' axonal projections in higher brain centres.
Through this elegant, iterative photolabelling approach, Ruta et al. could thus move stepwise through the brain to the output neurons that are likely to link directly with motor pathways. Although the circuit they define corresponds to just a small piece of the wiring diagram defined in the larger-scale anatomical studies4,5, Ruta et al.3 go one crucial step further by confirming that these cells are functionally connected. They accomplish this by recording cVA-evoked activity in each of the identified neurons — an impressive achievement deep in the tiny fly brain — and by demonstrating that this activity depends on the presence of intact circuit components upstream.
What do these findings reveal about the neural control of courtship? First, they offer a draft roadmap of a circuit underlying a complex animal behaviour. Although relatively simple neural circuits for reflexes (such as gill withdrawal in the marine slug Aplysia14 or the escape response in the fruitfly15) have been delineated, the FruM circuit is the most sophisticated to be mapped so thoroughly, sometimes down to single-neuron resolution. As the male courts his target female, these neurons must integrate diverse sensory information. Yet, as Ruta and co-workers show for the cVA response pathway, the circuit can be surprisingly shallow, with as few as four neurons potentially linking sensory input to motor output. The techniques and tools these studies3,4,5 introduce also make it feasible to test the functional contributions of individual subpopulations of FruM neurons to these behaviours in males and females.
In addition, these studies identify an unexpected number of new sexual dimorphisms in FruM neurons, including several cases in which certain groups of these cells are present only in the male or the female brain. They also detect hundreds of putatively distinct connections between the axons and dendrites of FruM neurons that are common to both sexes (Fig. 1). Although these anatomical (and physiological) observations do not establish a causal relationship between dimorphic wiring and behaviour, they indicate that widely distributed, although often subtle, gender-specific neural connectivity may account for the different behaviour of males and females. How FruM specifies the 'male' properties of this circuit during development remains a fascinating unanswered question.
Finally, it is notable that nearly all of these dimorphisms reside in central brain neurons. This suggests that males and females detect many of the same external signals but interpret them differently to produce distinct behavioural responses. Intriguingly, earlier work6 showed that courtship circuitry in the thorax of female flies also contains motor-neuron output pathways that can produce wing-evoked love songs when artificially stimulated. Thus, in fruitflies at least, the principal determinants underlying the distinct behaviour of males and females seem to reside in the mind.
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