Leveraging the unisexual brain: Investigating the neuronal circuits underlying sexual behaviours.
Lead Research Organisation:
University of Oxford
Department Name: Physiology Anatomy and Genetics
Abstract
The complex interplay between a male and female of a species during courtship is one of the most remarkable examples of sexually dimorphic behaviour in the animal kingdom. Upon identifying a suitable partner, Drosophila melanogaster males initiate an elaborate courtship ritual that culminates with copulation. Drosophila females do not actively court males, yet it is her response to the male's advances that determines whether mating will actually occur. These are complex decisions and behaviours controlled by the brain, but working with flies has the advantage of using a vast array of genetic tools that allows us to identify and manipulate relevant neurons in the brain. With these tools we can ask how does the brain differ between the sexes, and how might these differences explain the distinct behaviours of males and females that are critical for reproductive success?
We are interested in identifying neurons that control courtship behaviours, understanding how they are connected with one another, and how they work together to specify a behaviour that is unique to one gender. In particular, we have focused on understanding the role of neurons that express doublesex (dsx), a gene that is key for determining sex in Drosophila. These dsx+ neurons control male courtship behaviour and aspects of female receptivity. Yet little is known about the specific role of dsx+ neurons in the brain, which are believed to control mating decisions. We recently used genetic tools to manipulate the function of these neurons in the brain of females and males and evaluate the effects on sexual behaviour. Unexpectedly, when we artificially activated dsx+ neurons in the female brain, these females began to behave like males by displaying male-typical courtship behaviours towards other females or males.
This experiment demonstrates that both male and female brains have the necessary neurons capable of inducing male-specific behaviours, regardless of biological sex. So why don't females normally behave like males? And, what is different about the male and the female brain that allows them to behave in a sex-appropriate manner?
Using our assay to induce male-typical courtship behaviours in females we can now study sex differences in the brain that cause only males to exhibit these courtship behaviours. Specifically, we propose to address the following fundamental questions: (i) which specific neurons in the brain are capable of inducing male-typical courtship behaviours in females? (ii) how does their activity differ between sexes? (iii) Can we identify sex-differences in other neurons of the same circuitry that account for sexually dimorphic behaviours?
This study provides the opportunity to study how neurons operate differently to control mating decisions in females and males. Comparing the structure, function and activity of neurons in the brain of both sexes will reveal principles of brain organization that underlie unisex and sex-specific circuitry in flies. These studies offer great potential for understanding fundamental neuronal mechanisms that are present across species, and represent a key step toward understanding the complex inner workings of higher organisms.
We are interested in identifying neurons that control courtship behaviours, understanding how they are connected with one another, and how they work together to specify a behaviour that is unique to one gender. In particular, we have focused on understanding the role of neurons that express doublesex (dsx), a gene that is key for determining sex in Drosophila. These dsx+ neurons control male courtship behaviour and aspects of female receptivity. Yet little is known about the specific role of dsx+ neurons in the brain, which are believed to control mating decisions. We recently used genetic tools to manipulate the function of these neurons in the brain of females and males and evaluate the effects on sexual behaviour. Unexpectedly, when we artificially activated dsx+ neurons in the female brain, these females began to behave like males by displaying male-typical courtship behaviours towards other females or males.
This experiment demonstrates that both male and female brains have the necessary neurons capable of inducing male-specific behaviours, regardless of biological sex. So why don't females normally behave like males? And, what is different about the male and the female brain that allows them to behave in a sex-appropriate manner?
Using our assay to induce male-typical courtship behaviours in females we can now study sex differences in the brain that cause only males to exhibit these courtship behaviours. Specifically, we propose to address the following fundamental questions: (i) which specific neurons in the brain are capable of inducing male-typical courtship behaviours in females? (ii) how does their activity differ between sexes? (iii) Can we identify sex-differences in other neurons of the same circuitry that account for sexually dimorphic behaviours?
This study provides the opportunity to study how neurons operate differently to control mating decisions in females and males. Comparing the structure, function and activity of neurons in the brain of both sexes will reveal principles of brain organization that underlie unisex and sex-specific circuitry in flies. These studies offer great potential for understanding fundamental neuronal mechanisms that are present across species, and represent a key step toward understanding the complex inner workings of higher organisms.
Technical Summary
1-Identifying olfactory inputs inducing male-typical courtship behaviours in females
We will identify contributing olfactory pathways to male-typical courtship behaviours in brain-activated females by removing specific olfactory receptor genes, and assessing whether this results in the 'loss' of male-typical behaviours.
2- Mapping dsx+ neurons capable of inducing male-typical courtship behaviours in females
(i) We will parse brain dsx+ neurons into functionally distinct sub-populations using dsx alleles developed in the lab (dsxGAL4-DBD, dsxLexA and dsxGAL4) with established intersectional tools. (ii) We will carry out a thermogenetic screen to identify any dsx+ neurons in the female brain that, upon activation, triggers male-typical behaviours. (iii) Candidate lines will be neuroanatomically characterised using image brain registration to generate a functional map of dsx-brain neurons in males and females.
3- Characterising the physiological properties of dsx+ neurons in the brain
(i) Using optogenetics tools we will test different possibilities that could explain why females do not usually display male-typical courtship behaviours: (a) dsx+ neurons in females require higher thresholds to induce male-typical courtship behaviours than males. (b) dsx+ neurons in the brain are actively repressed by other neurons in females. (ii) We will monitor changes in the activity of candidate dsx+ neurons in live preparations of the nervous system of males and females using genetically encoded calcium indicators in the presence of fly odours.
4- Identifying neurons connected to dsx+ neurons in the brain
(i) We will carry out an in silico screen to identify upstream and downstream neurons connected to dsx+ neurons. (ii) We will select candidate lines displaying male-typical behaviours upon neuronal activation. (iii) We will test anatomical connectivity between dsx+ neurons and candidate neurons using GRASP and pre/post-synaptic markers in females and males.
We will identify contributing olfactory pathways to male-typical courtship behaviours in brain-activated females by removing specific olfactory receptor genes, and assessing whether this results in the 'loss' of male-typical behaviours.
2- Mapping dsx+ neurons capable of inducing male-typical courtship behaviours in females
(i) We will parse brain dsx+ neurons into functionally distinct sub-populations using dsx alleles developed in the lab (dsxGAL4-DBD, dsxLexA and dsxGAL4) with established intersectional tools. (ii) We will carry out a thermogenetic screen to identify any dsx+ neurons in the female brain that, upon activation, triggers male-typical behaviours. (iii) Candidate lines will be neuroanatomically characterised using image brain registration to generate a functional map of dsx-brain neurons in males and females.
3- Characterising the physiological properties of dsx+ neurons in the brain
(i) Using optogenetics tools we will test different possibilities that could explain why females do not usually display male-typical courtship behaviours: (a) dsx+ neurons in females require higher thresholds to induce male-typical courtship behaviours than males. (b) dsx+ neurons in the brain are actively repressed by other neurons in females. (ii) We will monitor changes in the activity of candidate dsx+ neurons in live preparations of the nervous system of males and females using genetically encoded calcium indicators in the presence of fly odours.
4- Identifying neurons connected to dsx+ neurons in the brain
(i) We will carry out an in silico screen to identify upstream and downstream neurons connected to dsx+ neurons. (ii) We will select candidate lines displaying male-typical behaviours upon neuronal activation. (iii) We will test anatomical connectivity between dsx+ neurons and candidate neurons using GRASP and pre/post-synaptic markers in females and males.
Planned Impact
Who will benefit from this research proposal?
Discoveries in Drosophila have greatly contributed to our understanding of neuroscience. An unequaled wealth of genetic techniques and strategies has permitted landmark discoveries in nervous system development and function. Such findings have generated and directed many research efforts in vertebrate neuroscience. After 100 years, Drosophila continues to be the choice model system for many neuroscientists. The combinational use of powerful research tools will ensure that this model organism will continue to lead to key discoveries that will impact vertebrate neuroscience. Moreover, working with Drosophila means less use of vertebrate models, saving on housing and husbandry costs as well as ethical considerations. This has long been a goal of the UK Research Councils and of society at large and falls under the aims of the 3Rs programme: replacement, reduction and refinement. We are using the Drosophila nervous system to study behavioural choices during courtship. Because the Drosophila nervous system is more accessible experimentally, and has fewer neurons than vertebrate brains, we believe it will yield insights into the mechanisms of behavioural choice that are much harder to study in higher vertebrates.
Moreover, discoveries that contribute to our understanding of reproductive behaviours in Drosophila are particularly important to other dipteran insects of medical importance. For example, mosquitoes and mosquito-borne diseases continue to plague mankind throughout the world. Of the critical behaviours that characterise the mosquito life strategy, mating is probably the least understood and most understudied. Yet, as mosquitoes depend on sexual reproduction for species maintenance, this aspect of mosquito biology should receive more attention when seeking new avenues for mosquito control and interventions for mosquito-borne disease. (see recent BBSRC highlight, http://www.bbsrc.ac.uk/news/people-skills-training/2014/140414-f-innovators-pt1-alphey.aspx)
How might individuals, organisations or society benefit from this research?
Sexually reproducing species often exhibit gender dimorphisms in behaviours such as courtship, aggression, and parental care. Defining the mechanisms underlying sexual differentiation of the brain and behaviour is one of biology's greatest challenges. Communicating how and why we use model organisms to study these basic mechanisms common to many forms of life is extremely important. S.F.G. has undertaken outreach activities with local schools, particularly during Science Week; presented his lab's work through Café Scientifique (www.cafescientifique.org/), a forum for debating science issues, which is committed to promoting public engagement with science and to making science accountable; and participated in the Channel4 BRITDOC-Festival, which brings together researchers, with some of the UK's foremost documentary filmmakers to exchange ideas and explore the potential for collaborations. This proposal has not only the potential for medically relevant discoveries but will also produce simple, yet provocative experimental paradigms, and discussion groups for teaching school children and undergraduates alike.
Discoveries in Drosophila have greatly contributed to our understanding of neuroscience. An unequaled wealth of genetic techniques and strategies has permitted landmark discoveries in nervous system development and function. Such findings have generated and directed many research efforts in vertebrate neuroscience. After 100 years, Drosophila continues to be the choice model system for many neuroscientists. The combinational use of powerful research tools will ensure that this model organism will continue to lead to key discoveries that will impact vertebrate neuroscience. Moreover, working with Drosophila means less use of vertebrate models, saving on housing and husbandry costs as well as ethical considerations. This has long been a goal of the UK Research Councils and of society at large and falls under the aims of the 3Rs programme: replacement, reduction and refinement. We are using the Drosophila nervous system to study behavioural choices during courtship. Because the Drosophila nervous system is more accessible experimentally, and has fewer neurons than vertebrate brains, we believe it will yield insights into the mechanisms of behavioural choice that are much harder to study in higher vertebrates.
Moreover, discoveries that contribute to our understanding of reproductive behaviours in Drosophila are particularly important to other dipteran insects of medical importance. For example, mosquitoes and mosquito-borne diseases continue to plague mankind throughout the world. Of the critical behaviours that characterise the mosquito life strategy, mating is probably the least understood and most understudied. Yet, as mosquitoes depend on sexual reproduction for species maintenance, this aspect of mosquito biology should receive more attention when seeking new avenues for mosquito control and interventions for mosquito-borne disease. (see recent BBSRC highlight, http://www.bbsrc.ac.uk/news/people-skills-training/2014/140414-f-innovators-pt1-alphey.aspx)
How might individuals, organisations or society benefit from this research?
Sexually reproducing species often exhibit gender dimorphisms in behaviours such as courtship, aggression, and parental care. Defining the mechanisms underlying sexual differentiation of the brain and behaviour is one of biology's greatest challenges. Communicating how and why we use model organisms to study these basic mechanisms common to many forms of life is extremely important. S.F.G. has undertaken outreach activities with local schools, particularly during Science Week; presented his lab's work through Café Scientifique (www.cafescientifique.org/), a forum for debating science issues, which is committed to promoting public engagement with science and to making science accountable; and participated in the Channel4 BRITDOC-Festival, which brings together researchers, with some of the UK's foremost documentary filmmakers to exchange ideas and explore the potential for collaborations. This proposal has not only the potential for medically relevant discoveries but will also produce simple, yet provocative experimental paradigms, and discussion groups for teaching school children and undergraduates alike.
Organisations
Publications
Allen AM
(2020)
A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord.
in eLife
Chiu H
(2021)
A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila.
in Cell
Flintham EO
(2018)
Interactions between the sexual identity of the nervous system and the social environment mediate lifespan in Drosophila melanogaster.
in Proceedings. Biological sciences
Goodwin S
(2018)
Editorial overview: Daylighting sex differences in physiology
in Current Opinion in Physiology
Mezzera C
(2020)
Ovipositor Extrusion Promotes the Transition from Courtship to Copulation and Signals Female Acceptance in Drosophila melanogaster.
in Current biology : CB
Neville MC
(2021)
Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster.
in Journal of neurogenetics
Nojima T
(2021)
A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior.
in Current biology : CB
Rezával C
(2016)
Activation of Latent Courtship Circuitry in the Brain of Drosophila Females Induces Male-like Behaviors.
in Current biology : CB
Rings A
(2019)
To court or not to court - a multimodal sensory decision in Drosophila males.
in Current opinion in insect science
| Description | Drosophila males, unlike females, exhibit an elaborate courtship display towards a potential mate. Sex-specific behaviours are hardwired into the nervous system via the actions of two key sex determination transcriptional regulators, doublesex (dsx) and fruitless (fru) (Pavlou and Goodwin, 2013). Activation of male-specific dsx/fru+ neurons in the brain has been shown to initiate courtship in males, suggesting that neural circuits only present in the male brain trigger such sex-specific behaviours. Surprisingly, in a screen to investigate the role of dsx neurons in the female brain, we found that artificial activation of a specific subset of neurons in the brain (dsx-pC1) induced Drosophila females to behave like males by inducing male-typical courtship behaviours. This behavioural change in activated females is dependent on the presence of another fly; allowing us to identify specific volatile pheromones present in both males and females that are able to promote male-courtship behaviours in these females. It has been proposed that the activity of male P1 neurons sets the threshold for the arousal state of males important for courtship initiation (Pan et al., 2012). Our experiments suggest that dsx-pC1 neurons in females might require higher thresholds (arousal levels) to induce male-courtship behaviours compared to males, which in turn could explain why females do not normally exhibit male behaviours. We have also shown that increasing levels of activation in dsx-pC1 neurons causes females to transition from female-specific behaviours (increased receptivity) to male-specific behaviours. As a consequence, wild-type males 'courted' by activated females cease their courtship display and, instead, avoid the courting female. Assuming no obvious change in pheromonal signals in these activated females, the switch in male behaviour from courtship to 'avoidance' is fascinating; procreation has switched to abstention. This opens up a new line of investigation, looking at a real-time male behavioural transition in response to conflicting sexual signals. While male courtship behaviour 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 behavioural program. |
| Exploitation Route | It was previously proposed that male courtship behaviour in Drosophila, such as song, may result from the activation of brain neurons that are unique to males. Here, we show that the female brain is capable of inducing male-like behaviours. Notably, a male-sexual behaviour effector circuit has been shown to be present in the brain of female mice, which is normally repressed. Hence, the existence of functional neuronal circuitry underlying male-specific behaviours in the normal female brain seems to be a conserved feature of courtship circuit organization in both invertebrates and vertebrates. |
| Sectors | Education |
| Description | The PI gave a public outreach lecture, part of a series of neuroscience lectures organized by Oxford Sparks (https://www.oxfordsparks.ox.ac.uk/content/sex-flies-and-videotape), to promote fundamental brain research. |
| First Year Of Impact | 2017 |
| Sector | Education |
| Impact Types | Cultural,Societal |
| Description | video release of our findings |
| Form Of Engagement Activity | Engagement focused website, blog or social media channel |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | video we made in house to publicise our findings from this grant |
| Year(s) Of Engagement Activity | 2016 |
| URL | https://vimeo.com/177551510 |