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Sexually dimorphic neuronal circuits underlying social behaviours in Drosophila

Periodic Reporting for period 1 - sexual dimorphism (Sexually dimorphic neuronal circuits underlying social behaviours in Drosophila)

Reporting period: 2018-09-01 to 2020-08-31

Males and females show significant differences in social behaviours, which depend on sex-specific neuronal organization. These differences are critical for reproduction, parenting and other basic interactions between animals. However, the neural circuitry underlying sexually dimorphic patterns is mostly unknown.
Sexually dimorphic behaviour in Drosophila is a key model system for revealing how genetically-determined properties of neuronal circuits define behaviour. Drosophila offers a unique model system to decipher the logic of dimorphic neuronal circuits, with its wealth of anatomical and neurogenetic tools, and a repertoire of dimorphic sexual behaviours. Although many sensory stimuli are integrated to regulate social behaviour, one of the best understood is the Drosophila male pheromone cVA, which promotes mating in females but repels other males and promotes inter-male aggression. Recent work from the host lab identified, for the first time, a sexually dimorphic switch in neuronal connectivity. The transcriptional master regulator fruitless rewires connections between pheromone responsive input neurons and two different target neuron populations in male and female brains. This study opened the question whether different target populations between males and females promote distinct behaviours. The current grant focused on three interdisciplinary aims that built on these results by establishing a causal role for specific wiring differences in regulating sexually dimorphic social behaviours; and studying how simple switches are assembled into more complex networks, at the interface of sensory processing and behavioural control.
The main research questions were how is the pheromone processing pathway organized in male and female brains, and how do differences in this pathway between male and female affect sexual behaviour. Understanding the logic of sexually dimorphic circuits also teaches us how simple circuit motifs are assembled into complex networks, from sensory input to behavioural output.
Conclusions of the action:
Using the emerging Drosophila connectome (Figure 1A), we reconstructed the neuroanatomical map of brain connections in the pheromone processing circuitry. This neuroanatomical wiring map spans from sensory neurons (olfactory, gustatory and mechanical) to descending neurons controlling motor output. Based on the wiring map we identified five novel sets of neurons in key points transmitting pheromone information. We then identified genetic driver lines for each of these subsets, tested the functionality of the connectivity using functional imaging, and manipulated these neuronal subsets in the brain while flies were freely behaving, in both sexes. We uncovered how each neuronal subset contributes to dimorphic social behaviours, including male-male aggression, male-female courtship, female receptivity, and female egg laying. Our work provides a direct link between anatomical differences and functional differences, and pinpoint the roles of specific circuit elements in regulating sexual behaviours.
Results overview:
1. cVA logic: a single cVA pheromone input diverges into parallel neuronal pathways, with distinct and sex-specific effects on sexual behaviour. in females, one pathway has privileged access to central neurons directly regulating mating behaviour (lvPN2 to pC1; see Figure 1B), while the other pathway allows further processing of the signal as it undergoes multisensory integration with gustatory channels (olfactory lPN and gustatory G2N-SLP1 neurons converge onto aSP-g neurons; see Figure 1B). Parallel pathways, direct and indirect, enable more flexible control and expression of distinct behaviours depending on social/environmental context.
2. Sensory integration: third order aSP-g neurons integrate taste and odour sensory information and bidirectionally modulate female receptivity. Functional imaging found supra-linear integration (synergy) when these neurons were presented with both taste and odour inputs.
3. Central convergence: downstream of aSP-g, layered axo-axonal connections leading to pC1 neurons sequentially gate different streams of sensory information; this gated hierarchy enables the animal to make a more reliable behavioural decision.
4. Activating each layer separately bidirectionally modulates sexual receptivity, but activating either aSP-g or ascending dMS6 neurons while blocking the downstream pC1 could not induce female receptivity, suggesting hierarchical integration by pC1.
5. Flexible behaviour choice: Multiple nodes in the circuit strongly modulate more than one behaviour, for example female receptivity AND aggression, or female receptivity AND egg-laying. These results demonstrate that a single node could participate in different networks and therefore serve for different purposes. For decoding function from brain activity, this result suggests that partial readout of neuronal activity is insufficient to understand the function of a circuitry. One component may participate in multiple functions, perhaps depending on animal state, governed by social or internal context.
6. We found that axo-axonic connections can have a net excitation effect, detectable even at a distant soma, suggesting a wider range of computations possible by axonal inputs than previously thought.

Exploitation and dissemination:
These results had been presented in multiple international and local meetings. These results are currently being prepared into a manuscript which will be submitted to a peer reviewed Open Access journal.
We combined state-of-the-art methods including connectomics, physiology and behaviour analyses guided by artificial intelligence to pinpoint commonalities and differences in neuronal organization between male and female fly brains, providing direct links from brain morphology to function. This project uncovered in both sexes how multiple dimorphic elements are assembled into a circuit extending from sensory periphery to descending output, and established the relevance of these components for dimorphic behaviour. The discoveries made during the grant have wider implications on our understanding of neuronal circuit logic, specifically in context of sex differences, for example the extent to which circuit elements are conserved or differentiated between sexes, and their role in regulating sexual behaviours. These findings capture exciting novel network motifs, for example demonstrating the role of similar circuit elements in different behaviours; the logic of hierarchical mutli-sensory integration for robust decision making, and novel neuronal computations such as axo-axonal excitatory connections on inhibitory neurons, turning a plus sign signal into a minus.

Additionally, our research topic can be used to support societal discussion on gender equality and diversity, as part of the European Commission goals. The topic of gender differences is socially relevant and attractive for discussions with general public. The researcher is committed to advocate gender equality and diversity and has been actively doing so across multiple science communication platforms.
The project has provided extensive training opportunities for the researcher, and enabled further funding opportunities for the researcher and the host lab. Progress due to the project: two manuscripts are in the process of being written and submitted to top peer-reviewed international journals.
Figure 1