Dispatch R243
Aggression: Tachykinin Is All the Rage Animals are constantly receiving information about their environment that must be filtered to ensure that they respond in the appropriate manner. New data have revealed how neurons in male Drosophila promote a heightened state of aggression in response to a rival male. Hania J. Pavlou, Megan C. Neville, and Stephen F. Goodwin* Why fight? What advantage does it give you? When and where do you do it? Who do you do it with? Even during the relatively short life-span of the fruit fly Drosophila melanogaster these questions and behaviors are relevant. Aggressive acts between members of the same species are most often a result of competitive access to one of two key resources: food or sex (a potential mate). Aggression in response to these resources tends to vary dramatically depending on the gender of the animal, with males often making more overt behavioral displays. In Drosophila, males and females both display aggression, though the manner in which they respond to a potential foe and the vigour with which they fight differs between them [1]. External visual, auditory, olfactory and gustatory cues, to name a few, will all feed into how a fly responds to a potential rival. Internal physiological cues will also feed into how a fly responds—for example, the state of hunger might directly influence the fly’s motivation to fight over a source of food. The additional innate drive to reproduce along with other factors such as age and experience will further influence the male’s desire to fight for a mate. Thus, the underlying impetus, or motivation, to act aggressively depends on a complex interplay between all of these cues, internal and external, as well as an ability to mitigate behavior in different contexts. Despite advances in our ability to dissect the neural circuitry underlying various innate behaviors, relatively little detail is known about the circuitry that specifies aggression. In a recent paper, Asahina et al. [2] explored the neural basis of aggression by investigating the contribution of neuropeptide releasing neurons. By combining thermogenetic activation of neurons — using the thermosensitive cation channel TRPA1 which increases neuronal excitability in
response to heat — with the ability to genetically target peptidergic neurons, the authors performed a behavioral screen to identify neurons that, when activated, caused an increase in aggressive behaviors. The screen successfully identified two peptidergic lines that showed dramatically high levels of inter-male aggression, both of which were associated with the Tachykinin gene (Tk). Remarkably, Tk encodes the Drosophila homologue of Substance P, a molecule that has been implicated in aggression in a number of mammalian species [3]. A closer anatomical look at the aggression-inducing neurons revealed a striking sexual dimorphism; the presence of a small cluster of Tk+ lateral protocerebral neurons in the male brain only. The analysis was refined by intersecting the expression of Tk with that of the gene fruitless (fru), a key regulator of male-specific behaviors [4]. This approach showed an overlap in expression between Tk and the male-specific form of fru (Tk+/fru+) in a subset of the Tk+ neurons. Activation of only the Tk+/fru+ neurons again resulted in high levels of inter-male aggression, while genetically silencing these same neurons significantly reduced, but did not abolish, inter-male aggression (Figure 1A). As fru+ neurons are known to play key roles in most, if not all, aspects of male-specific courtship behaviors [4], it was important to establish if these Tk+/fru+ neurons also played a role in courtship behavior. Interestingly, when Tk+/fru+ neurons were activated in males in the presence of a female, the males did not attempt to fight the female but rather courted her (Figure 1B). This supports the view that courtship and aggressive behaviors are neuroanatomically separable [5], and suggests that sensory signals from the female either directly inhibit the aggressive response and/or the elicitation of courtship behavior over-rides it.
Asahina et al. [2] broadened their study by examining the role of Tachykinin itself in promoting aggressive behaviors, and more specifically its role in the identified neurons of the male brain. They generated two novel null alleles of Tk, both of which were viable and exhibited no obvious locomotion or courtship defects. When inter-male aggression was examined, however, these mutants exhibited significant deficits in the display of this behaviour. Aggression in these mutants was not abolished, demonstrating that Tk is important but not necessary for the display of aggression. When Tk+/fru+ neurons were thermogenetically activated in a Tk-null mutant background, elevated levels of aggression were still observed, but not at the intensity seen when Tachykinin is present, suggesting that there are additional aggression-inducing signals released by these neurons. It is common for neuropeptides to be co-released with small-molecule neurotransmitters; it is perhaps the release of such molecules that contribute to this residual effect. To address this, the authors present evidence that the identified Tk+ neurons are cholinergic, which is consistent with previous work that implicated cholinergic neurons in sexually dimorphic aggression [6]. However, establishing a clear role for this or other neurotransmitters in these neurons will require further investigation. Asahina et al. [2] next examined the role of the known cognate receptors of Tachykinin, Takr86C and Takr99D, in aggression. Both a novel null mutant of Takr86C (generated in this study) and a putative loss-of-function Takr99D mutant displayed normal levels of aggression; however, a double mutant showed decreased levels of aggression. The complex relationship between Tachykinin and its receptors was revealed when activation of Tk+ neurons only in a Takr86-null background was capable of supressing levels of induced aggression. Further experiments are required to determine the specific roles of these receptors in mediating aggression. So what is the mechanism by which the critical Tk+/fru+ cluster specifically promotes inter-male aggression? Does the release of Tachykinin from these neurons regulate the flow of sensory
Current Biology Vol 24 No 6 R244
A
Tk+/fru+
B
Tk+/fru+
In presence of another male
Tk+/fru+
Tk+/fru+
In presence of a female C or
Tk+/fru+
In presence of a male & female Current Biology
Figure 1. A cluster of Tk+/fru+ neurons in the male brain promotes inter-male aggression. (A) In the presence of a male, activation of Tk+/fru+ neurons establishes a heightened state of aggressive arousal, leading to an increased display of aggressive behaviours. Unilateral grey Tk+/fru+ neurons in the male hemi-brain signify an inactive state while unilateral red Tk+/fru+ neurons signify an active state. (B) In the presence of a female, activation of Tk+/fru+ neurons does not lead to aggression towards the female, rather male courtship behaviour towards the female is observed. (C) Depiction of a state of conflict to pose the question: when Tk+/fru+ neurons are activated which behaviour, aggression or courtship, is preferentially displayed in the presence of both males and females?
information by altering the dynamics of ‘Tachykinin receptor’-expressing neurons? To tackle this, Asahina et al. [2] first reasoned that if Tachykinin serves to establish a ‘heightened state of response’ (more likely to fight), males would successfully display aggressive behaviors in the absence of known stimulatory cues. Individual sensory cues known to enhance male aggression were removed while Tk+/fru+ neurons were activated.
Interestingly, the removal of any one cue did not eliminate the increase in inter-male aggression; however, specific cues, such as food, did decrease the overall levels of activated aggression. These data not only demonstrate that Tachykinin modulates the excitability and/or sensitivity of ‘Tachykinin receptor’expressing neurons, but also show that the effects of Tachykinin modulation occur downstream of sensory processing. Neuromodulators like Tachykinin have long been known to modulate adaptive animal behaviors and physiological processes. This enables the integration of all external sensory and internal physiological cues that are received at any given point in time, and the appropriate interpretation of this information to ensure the generation of the suitable behavioral response. By moving away from the context of lab-based impoverished environments, the real importance of this work will come to light when males are presented with both males and females in a more naturalistic assay [7]. As courtship behaviors and aggression are largely mutually exclusive responses that are displayed by the male fly, what takes precedence in this context and what factors influence this ‘decision’ (Figure 1C)? Asahina et al. [2] postulate that the Tk+/fru+ cluster may be equivalent to previously mapped fru-expressing third-order olfactory neurons [8,9]. Intriguingly, previous work has shown that these neurons respond to the male pheromone 11-cis-vaccenyl acetate (cVA) [10,11]. cVA has been shown to promote aggression and supress courtship in males, as well as increase receptivity in females [12]; the mechanism by which these neurons respond to cVA and elicit sex-specific behaviors depends on their sexually dimorphic wiring [11]. Assuming the Tk+/fru+ cluster is in fact a subset of this cVA-responsive cluster, it is conceivable that cVA would serve as a prominent trigger for male-specific activation of Tk+/fru+ neurons and subsequent Tachykinin release, leading to a heightened state of arousal. Future studies focusing on the identification and characterisation of neurons that express the cognate
receptors of Tachykinin will begin to elucidate the mechanism by which Tachykinin modulates the behavioral salience of the male fly. The localization of both upstream and downstream components may hint at the essential circuit elements that underlie male aggression; however, ultimately identifying the changes to their activity in response to Tachykinin release and establishing how this response relates to aggression will surely be the focus of studies for many years to come. References 1. Ferna´ndez, M.P., and Kravitz, E.A. (2013). Aggression and courtship in Drosophila: pheromonal communication and sex recognition. J. Comp. Physiol. A 199, 1065–1076. 2. Asahina, K., Watanabe, K., Duistermars, B.J., Hoopfer, E., Gonza´lez, C.R., Eyjo´lfsdo´ttir, E.A., Perona, P., and Anderson, D.J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156, 221–235. 3. Katsouni, E., Sakkas, P., Zarros, A., Skandali, N., and Liapi, C. (2009). The involvement of substance P in the induction of aggressive behavior. Peptides 30, 1586–1591. 4. Yamamoto, D., and Koganezawa, M. (2013). Genes and circuits of courtship behaviour in Drosophila males. Nat. Rev. Neurosci. 14, 681–692. 5. Chan, Y.B., and Kravitz, E.A. (2007). Specific subgroups of FruM neurons control sexually dimorphic patterns of aggression in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 104, 19577–19582. 6. Mundiyanapurath, S., Chan, Y.B., Leung, A.K., and Kravitz, E.A. (2009). Feminizing cholinergic neurons in a male Drosophila nervous system enhances aggression. Fly 3, 179–184. 7. Taghert, P.H., and Nitabach, M.N. (2012). Peptide neuromodulation in invertebrate model systems. Neuron 76, 82–97. 8. Cachero, S., Ostrovsky, A.D., Yu, J.Y., Dickson, B.J., and Jefferis, G.S.X.E. (2010). Sexual dimorphism in the fly brain. Curr. Biol. 20, 1589–1601. 9. Yu, J.Y., Kanai, M.I., Demir, E., Jefferis, G.S.X.E., and Dickson, B.J. (2010). Cellular organization of the neural circuit that drives Drosophila courtship behavior. Curr. Biol. 20, 1602–1614. 10. Ruta, V., Datta, S.R., Vasconcelos, M.L., Freeland, J., Looger, L.L., and Axel, R. (2010). A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468, 686–690. 11. Kohl, J., Ostrovsky, A.D., Frechter, S., and Jefferis, G.S. (2013). A bidirectional circuit switch reroutes pheromone signals in male and female brains. Cell 155, 1610–1623. 12. Kurtovic, A., Widmer, A., and Dickson, B.J. (2007). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542–546.
Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2014.02.017
Aggression: Tachykinin Is All the Rage Animals are constantly receiving information about their environment that must be filtered to ensure that they respond in the appropriate manner. New data have revealed how neurons in male Drosophila promote a heightened state of aggression in response to a rival male. Hania J. Pavlou, Megan C. Neville, and Stephen F. Goodwin* Why fight? What advantage does it give you? When and where do you do it? Who do you do it with? Even during the relatively short life-span of the fruit fly Drosophila melanogaster these questions and behaviors are relevant. Aggressive acts between members of the same species are most often a result of competitive access to one of two key resources: food or sex (a potential mate). Aggression in response to these resources tends to vary dramatically depending on the gender of the animal, with males often making more overt behavioral displays. In Drosophila, males and females both display aggression, though the manner in which they respond to a potential foe and the vigour with which they fight differs between them [1]. External visual, auditory, olfactory and gustatory cues, to name a few, will all feed into how a fly responds to a potential rival. Internal physiological cues will also feed into how a fly responds—for example, the state of hunger might directly influence the fly’s motivation to fight over a source of food. The additional innate drive to reproduce along with other factors such as age and experience will further influence the male’s desire to fight for a mate. Thus, the underlying impetus, or motivation, to act aggressively depends on a complex interplay between all of these cues, internal and external, as well as an ability to mitigate behavior in different contexts. Despite advances in our ability to dissect the neural circuitry underlying various innate behaviors, relatively little detail is known about the circuitry that specifies aggression. In a recent paper, Asahina et al. [2] explored the neural basis of aggression by investigating the contribution of neuropeptide releasing neurons. By combining thermogenetic activation of neurons — using the thermosensitive cation channel TRPA1 which increases neuronal excitability in
response to heat — with the ability to genetically target peptidergic neurons, the authors performed a behavioral screen to identify neurons that, when activated, caused an increase in aggressive behaviors. The screen successfully identified two peptidergic lines that showed dramatically high levels of inter-male aggression, both of which were associated with the Tachykinin gene (Tk). Remarkably, Tk encodes the Drosophila homologue of Substance P, a molecule that has been implicated in aggression in a number of mammalian species [3]. A closer anatomical look at the aggression-inducing neurons revealed a striking sexual dimorphism; the presence of a small cluster of Tk+ lateral protocerebral neurons in the male brain only. The analysis was refined by intersecting the expression of Tk with that of the gene fruitless (fru), a key regulator of male-specific behaviors [4]. This approach showed an overlap in expression between Tk and the male-specific form of fru (Tk+/fru+) in a subset of the Tk+ neurons. Activation of only the Tk+/fru+ neurons again resulted in high levels of inter-male aggression, while genetically silencing these same neurons significantly reduced, but did not abolish, inter-male aggression (Figure 1A). As fru+ neurons are known to play key roles in most, if not all, aspects of male-specific courtship behaviors [4], it was important to establish if these Tk+/fru+ neurons also played a role in courtship behavior. Interestingly, when Tk+/fru+ neurons were activated in males in the presence of a female, the males did not attempt to fight the female but rather courted her (Figure 1B). This supports the view that courtship and aggressive behaviors are neuroanatomically separable [5], and suggests that sensory signals from the female either directly inhibit the aggressive response and/or the elicitation of courtship behavior over-rides it.
Asahina et al. [2] broadened their study by examining the role of Tachykinin itself in promoting aggressive behaviors, and more specifically its role in the identified neurons of the male brain. They generated two novel null alleles of Tk, both of which were viable and exhibited no obvious locomotion or courtship defects. When inter-male aggression was examined, however, these mutants exhibited significant deficits in the display of this behaviour. Aggression in these mutants was not abolished, demonstrating that Tk is important but not necessary for the display of aggression. When Tk+/fru+ neurons were thermogenetically activated in a Tk-null mutant background, elevated levels of aggression were still observed, but not at the intensity seen when Tachykinin is present, suggesting that there are additional aggression-inducing signals released by these neurons. It is common for neuropeptides to be co-released with small-molecule neurotransmitters; it is perhaps the release of such molecules that contribute to this residual effect. To address this, the authors present evidence that the identified Tk+ neurons are cholinergic, which is consistent with previous work that implicated cholinergic neurons in sexually dimorphic aggression [6]. However, establishing a clear role for this or other neurotransmitters in these neurons will require further investigation. Asahina et al. [2] next examined the role of the known cognate receptors of Tachykinin, Takr86C and Takr99D, in aggression. Both a novel null mutant of Takr86C (generated in this study) and a putative loss-of-function Takr99D mutant displayed normal levels of aggression; however, a double mutant showed decreased levels of aggression. The complex relationship between Tachykinin and its receptors was revealed when activation of Tk+ neurons only in a Takr86-null background was capable of supressing levels of induced aggression. Further experiments are required to determine the specific roles of these receptors in mediating aggression. So what is the mechanism by which the critical Tk+/fru+ cluster specifically promotes inter-male aggression? Does the release of Tachykinin from these neurons regulate the flow of sensory
Current Biology Vol 24 No 6 R244
A
Tk+/fru+
B
Tk+/fru+
In presence of another male
Tk+/fru+
Tk+/fru+
In presence of a female C or
Tk+/fru+
In presence of a male & female Current Biology
Figure 1. A cluster of Tk+/fru+ neurons in the male brain promotes inter-male aggression. (A) In the presence of a male, activation of Tk+/fru+ neurons establishes a heightened state of aggressive arousal, leading to an increased display of aggressive behaviours. Unilateral grey Tk+/fru+ neurons in the male hemi-brain signify an inactive state while unilateral red Tk+/fru+ neurons signify an active state. (B) In the presence of a female, activation of Tk+/fru+ neurons does not lead to aggression towards the female, rather male courtship behaviour towards the female is observed. (C) Depiction of a state of conflict to pose the question: when Tk+/fru+ neurons are activated which behaviour, aggression or courtship, is preferentially displayed in the presence of both males and females?
information by altering the dynamics of ‘Tachykinin receptor’-expressing neurons? To tackle this, Asahina et al. [2] first reasoned that if Tachykinin serves to establish a ‘heightened state of response’ (more likely to fight), males would successfully display aggressive behaviors in the absence of known stimulatory cues. Individual sensory cues known to enhance male aggression were removed while Tk+/fru+ neurons were activated.
Interestingly, the removal of any one cue did not eliminate the increase in inter-male aggression; however, specific cues, such as food, did decrease the overall levels of activated aggression. These data not only demonstrate that Tachykinin modulates the excitability and/or sensitivity of ‘Tachykinin receptor’expressing neurons, but also show that the effects of Tachykinin modulation occur downstream of sensory processing. Neuromodulators like Tachykinin have long been known to modulate adaptive animal behaviors and physiological processes. This enables the integration of all external sensory and internal physiological cues that are received at any given point in time, and the appropriate interpretation of this information to ensure the generation of the suitable behavioral response. By moving away from the context of lab-based impoverished environments, the real importance of this work will come to light when males are presented with both males and females in a more naturalistic assay [7]. As courtship behaviors and aggression are largely mutually exclusive responses that are displayed by the male fly, what takes precedence in this context and what factors influence this ‘decision’ (Figure 1C)? Asahina et al. [2] postulate that the Tk+/fru+ cluster may be equivalent to previously mapped fru-expressing third-order olfactory neurons [8,9]. Intriguingly, previous work has shown that these neurons respond to the male pheromone 11-cis-vaccenyl acetate (cVA) [10,11]. cVA has been shown to promote aggression and supress courtship in males, as well as increase receptivity in females [12]; the mechanism by which these neurons respond to cVA and elicit sex-specific behaviors depends on their sexually dimorphic wiring [11]. Assuming the Tk+/fru+ cluster is in fact a subset of this cVA-responsive cluster, it is conceivable that cVA would serve as a prominent trigger for male-specific activation of Tk+/fru+ neurons and subsequent Tachykinin release, leading to a heightened state of arousal. Future studies focusing on the identification and characterisation of neurons that express the cognate
receptors of Tachykinin will begin to elucidate the mechanism by which Tachykinin modulates the behavioral salience of the male fly. The localization of both upstream and downstream components may hint at the essential circuit elements that underlie male aggression; however, ultimately identifying the changes to their activity in response to Tachykinin release and establishing how this response relates to aggression will surely be the focus of studies for many years to come. References 1. Ferna´ndez, M.P., and Kravitz, E.A. (2013). Aggression and courtship in Drosophila: pheromonal communication and sex recognition. J. Comp. Physiol. A 199, 1065–1076. 2. Asahina, K., Watanabe, K., Duistermars, B.J., Hoopfer, E., Gonza´lez, C.R., Eyjo´lfsdo´ttir, E.A., Perona, P., and Anderson, D.J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156, 221–235. 3. Katsouni, E., Sakkas, P., Zarros, A., Skandali, N., and Liapi, C. (2009). The involvement of substance P in the induction of aggressive behavior. Peptides 30, 1586–1591. 4. Yamamoto, D., and Koganezawa, M. (2013). Genes and circuits of courtship behaviour in Drosophila males. Nat. Rev. Neurosci. 14, 681–692. 5. Chan, Y.B., and Kravitz, E.A. (2007). Specific subgroups of FruM neurons control sexually dimorphic patterns of aggression in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 104, 19577–19582. 6. Mundiyanapurath, S., Chan, Y.B., Leung, A.K., and Kravitz, E.A. (2009). Feminizing cholinergic neurons in a male Drosophila nervous system enhances aggression. Fly 3, 179–184. 7. Taghert, P.H., and Nitabach, M.N. (2012). Peptide neuromodulation in invertebrate model systems. Neuron 76, 82–97. 8. Cachero, S., Ostrovsky, A.D., Yu, J.Y., Dickson, B.J., and Jefferis, G.S.X.E. (2010). Sexual dimorphism in the fly brain. Curr. Biol. 20, 1589–1601. 9. Yu, J.Y., Kanai, M.I., Demir, E., Jefferis, G.S.X.E., and Dickson, B.J. (2010). Cellular organization of the neural circuit that drives Drosophila courtship behavior. Curr. Biol. 20, 1602–1614. 10. Ruta, V., Datta, S.R., Vasconcelos, M.L., Freeland, J., Looger, L.L., and Axel, R. (2010). A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468, 686–690. 11. Kohl, J., Ostrovsky, A.D., Frechter, S., and Jefferis, G.S. (2013). A bidirectional circuit switch reroutes pheromone signals in male and female brains. Cell 155, 1610–1623. 12. Kurtovic, A., Widmer, A., and Dickson, B.J. (2007). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542–546.
Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK. *E-mail: [email protected]
http://dx.doi.org/10.1016/j.cub.2014.02.017
