Birdsong consists of species-specific learned vocal sequences that are used primarily to attract mates and to repel competitors during the breeding season. However, many birds continue to sing at times when vocal production has no immediate or obvious impact on conspecific behavior. The mechanisms that ensure that animals produce important behaviors in contexts in which the function of these behaviors is not immediate or obvious are not known. One possibility is that animals engage in such behaviors because they are associated with pleasure. Here we examined the hypothesis that male European starlings sing outside of the breeding season in part because the act of singing in this context is facilitated and/or maintained by opioid-mediated reward. We measured song-associated reward using a conditioned place preference (CPP) test in male starlings producing fall, non-breeding season-typical song. We used quantitative real time PCR to measure expression of the enkephalin opioid precursor preproenkephalin (PENK) and mu opioid receptors (MOR) in the medial preoptic nucleus (POM; a region in which opioids are implicated in both reward and starling fall song) and additionally the song control region HVC as a control. Starlings developed a strong preference for a place that had been paired previously with the act of producing fall-typical song, indicating that fall song production was associated with a positive affective state. Both PENK and MOR mRNA expression in the POM, but not HVC, correlated positively with both individual reward state (as reflected in CPP) and undirected singing behavior. These results suggest that singing induces opioid receptor and enkephalin expression in the POM and consequent reward, and/or that opioid release in the POM induced by individual or environmental factors (e.g., the presence of food, safety of a flock or the absence of predators) induces a positive affective state which then facilitates singing behavior.

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Vocalizations coordinate social interactions in many species and often are important for behaviors such as mate attraction or territorial defense. Although the neural circuitry underlying vocal communication is well-known for some species, such as songbirds, the motivational processes that regulate vocal signals are not as clearly understood. Neurotensin (NT) is a neuropeptide implicated in motivation that can modulate the activity of dopaminergic neurons. Dopaminergic projections from the ventral tegmental area (VTA) are key to mediating highly motivated, goal-directed behaviors, including sexually-motivated birdsong. However, the role of NT in modifying vocal communication or other social behaviors has not been well-studied. Here in European starlings (Sturnus vulgaris) we analyzed relationships between sexually-motivated song and NT and NT1 receptor (NTSR1) expression in VTA. Additionally, we examined NT and NTSR1 expression in four regions that receive dopaminergic projections from VTA and are involved in courtship song: the medial preoptic nucleus (POM), the lateral septum (LS), Area X, and HVC. Relationships between NT and NTSR1 expression and non-vocal courtship and agonistic behaviors were also examined. NT expression in Area X positively related to sexuallymotivated song production. NT expression in POM positively correlated with non-vocal courtship behavior and agonistic behavior. NT expression in POM was greatest in males owning nesting sites, and the opposite pattern was observed for NTSR1 expression in LS. These results are the first to implicate NT in Area X in birdsong, and further highlight NT as a potential neuromodulator for the control of vocal communication and other social behaviors.

Converging data in songbirds support a central role for the medial preoptic nucleus (POM) in motivational aspects of vocal production. Recent data suggest that dopamine in the POM plays a complex modulatory role in the production of sexually-motivated song and that an optimal level of dopamine D1 receptor stimulation is required to facilitate singing behavior. To further explore this possibility, we used quantitative real time PCR to examine relationships between mRNA expression of D1 as well as D2 receptors in the POM (and also the lateral septum and Area X) and sexually-motivated singing behavior in male European starlings. Results showed that both males with the highest and lowest D1 expression in the POM sang significantly less than males with intermediate levels of expression. Furthermore, singing behavior rose linearly in association with increasing levels of D1 expression in POM but dropped abruptly, such that individuals with D1 expression values higher than the mean sang very little. Analysis of birds with low and intermediate levels of D1 expression in POM revealed strong positive correlations between D1 expression and song but negative relationships between D2 receptor expression and song. These findings support prior work suggesting an optimal level of POM D1 receptor stimulation best facilitates sexually-motivated singing behavior. Results also suggest that D2 receptors may work in opposition to D1 receptors in POM to modify vocal production.

Many species modify behavior in response to changes in resource availability or social status; however, the neural mechanisms underlying these modifications are not well understood. Prior work in male starlings demonstrates that status-appropriate changes in behavior involve brain regions that regulate social behavior and vocal production. Endocannabinoids are ubiquitously distributed neuromodulators that are proposed to play a role in adjusting behavior to match social status. As an initial step to provide insight into this hypothesis we observed flocks of male starlings in outdoor aviaries during the breeding season. We used quantitative real-time PCR to measure expression of endocannabinoid CB1 receptors in brain regions involved in social behavior and motivation (lateral septum [LS], ventral tegmental area [VTA], medial preoptic nucleus [POM]) and vocal behavior (Area X and robust nucleus of the arcopallium; RA). Males with nesting sites sang to females and displaced other males more than males without nesting sites. They also had higher levels of CB1 receptor expression in LS and RA. CB1 expression in LS correlated positively with agonistic behaviors. CB1 expression in RA correlated positively with singing behavior. CB1 in VTA also correlated positively with singing when only singing birds were considered. These correlations nicely map onto the well-established role of LS in agonistic behavior and the known role of RA in song production and VTA in motivation and song production. Studies are now needed to precisely characterize the role of CB1 receptors in these regions in the production of status-appropriate social behaviors.

Keywords: aggressionCardinalis cardinalis corticosterone female incubation maternal aggression nest defence northern cardinal testosterone Testosterone's (T) influence on male aggression has been well established in many vertebrate species, but the impact of T on female aggressive behaviour is poorly understood. Among birds, a link between T and female aggression is plausible, as females of many species exhibit a seasonal peak in T concentrations at the onset of breeding when social instability is greatest and they may have circulating T through much of the breeding season. However, investigations examining the relationship between T and female aggression are few and have yielded conflicting results, with experimentally or endogenously elevated T supporting aggressive behaviour in females of some species but not others, and T elevating with aggression at some points of the reproductive cycle but not others. We examined the relationship between endogenous levels of T and female aggression in the northern cardinal, Cardinalis cardinalis, a resident temperate species in which pairs exhibit prolonged territoriality and females have measurable levels of T year-round, including all stages of reproduction (incubation, nestling feeding, etc.). Using simulated nest intrusions, we assessed aggressive responses of incubating females to intrasexual 'intruders' at the nest and quantified T levels after each aggressive encounter. Displays of aggression towards 'intruders' varied among females; yet, individuals showing greater levels of aggression did not demonstrate higher levels of T. These results imply that T might not support maternal aggression in this species.