Male European Starlings Research Articles

Dietary fatty acids and flight-training influence the expression of the eicosanoid hormone prostacyclin in songbirds

Diet shifts can alter tissue fatty acid composition in birds, which is subsequently related to metabolic patterns. Eicosanoids, short-lived fatty acid-derived hormones, have been proposed to mediate these relationships but neither baseline concentrations nor the responses to diet and exercise have been measured in songbirds. We quantified a stable derivative of the vasodilatory eicosanoid prostacyclin in the plasma of male European Starlings (Sturnus vulgaris, N = 25) fed semisynthetic diets with either high (PUFA) or low (MUFA) amounts of n6 fatty acid precursors to prostacyclin. Plasma samples were taken from each bird before, immediately after, and two days following a 15-day flight-training regimen that a subset of birds (N = 17) underwent. We found elevated prostacyclin levels in flight-trained birds fed the PUFA diet compared to those fed the MUFA diet and a positive relationship between prostacyclin and body condition, indexed by fat score. Prostacyclin concentrations also significantly decreased at the final time point. These results are consistent with the proposed influences of precursor availability (i.e., dietary fatty acids) and regulatory feedback associated with exercise (i.e., fuel supply and inflammation), and suggest that prostacyclin may be an important mediator of dietary influence on songbird physiology.

Distinct patterns of activity within columns of the periaqueductal gray are associated with functionally distinct birdsongs.

Male songbirds produce female-directed songs in spring that convey a state of sexual motivation. Many songbirds also sing in fall flocks in affiliative/gregarious contexts in which song is linked to an intrinsic positive affective state. The periaqueductal gray (PAG) in mammals, which is organized into functional columns, integrates information from multiple brain regions and relays this information to vocal motor areas so that an animal emits a vocal signal reflective of its affective state. Here, we test the hypothesis that distinct columns in the songbird PAG play roles in the distinct affective states communicated by sexually motivated and gregarious song. We quantified the numbers of immediate early gene ZENK-positive cells in 16 PAG subregions in male European starlings (Sturnus vulgaris) after singing gregarious or sexually motivated song. Results suggest that distinct PAG columns in songbirds context-specifically regulate song, agonistic, and courtship behaviors. A second exploratory, functional tract-tracing study also demonstrated that inputs to the PAG from specific subregions of the medial preoptic nucleus may contribute to gregarious song and behaviors indicative of social dominance. Together, findings suggest that conserved PAG columns and inputs from the preoptic nucleus may play a role in context-specific vocal and other social behaviors.

The motivation to flock correlates with vocal-social behaviors and dopamine-related gene expression in male European starlings

It is proposed that songbird flocks are partly reinforced by positive social interactions, however not all flock mate interactions are positive. The combination of both positive and negative social interactions with flock mates may play a role in the motivation for birds to flock. The nucleus accumbens (NAc), medial preoptic area (POM), and ventral tegmental area (VTA) are implicated in vocal-social behaviors in flocks, including singing. Dopamine (DA) within these regions modifies motivated, reward-directed behaviors. Here, we begin to test the hypothesis that individual social interactions and DA within these regions are involved in the motivation to flock. Vocal-social behaviors were recorded in eighteen male European starlings in mixed-sex flocks in fall, when starlings are highly social and form large flocks. Males were then singly removed from their flock and the motivation to flock was quantified as the amount of time spent attempting to join a flock following separation. We used quantitative real-time polymerase chain reaction to measure expression of DA-related genes in the NAc, POM, and VTA. Birds producing high levels of vocal behaviors were more highly motivated to flock and had higher tyrosine hydroxylase (the rate-limiting enzyme in DA synthesis) expression in the NAc and VTA. Birds that received high levels of agonistic behaviors were less motivated to flock and had higher DA receptor subtype 1 expression in the POM. Overall, our findings suggest that interplay between social experience and DA activity in NAc, POM, and VTA plays a key role in social motivation in flocking songbirds.

Dehydroepiandrosterone (DHEA) increases undirected singing behavior and alters dopaminergic regulation of undirected song in non-breeding male European starlings (Sturnus vulgaris).

It has been proposed that in species that defend territories across multiple life history stages, brain metabolism of adrenal dehydroepiandrosterone (DHEA) regulates aggressive behavior at times when gonadal androgen synthesis is low (i.e. the non-breeding season). To date, a role for DHEA in the regulation of other forms of social behavior that are expressed outside of the context of breeding remains unknown. In this experiment, we used the European starling (Sturnus vulgaris) model system to investigate a role for DHEA in the neuroendocrine regulation of singing behavior by males in non-breeding condition. Starling song in a non-breeding context is spontaneous, not directed towards conspecifics, and functions to maintain cohesion of overwintering flocks. Using within-subjects design, we found that DHEA implants significantly increase undirected singing behavior by non-breeding condition male starlings. Given that DHEA is known to modulate multiple neurotransmitter systems including dopamine (DA) and DA regulates undirected song, we subsequently used immunohistochemistry for phosphorylated tyrosine hydroxylase (pTH, the active form of the rate-limiting enzyme in DA synthesis) to investigate the effect of DHEA on dopaminergic regulation of singing behavior in a non-breeding context. Pearson correlation analysis revealed a positive linear association between undirected singing behavior and pTH immunoreactivity in the ventral tegmental area and midbrain central gray of DHEA-implanted, but not control-implanted, males. Taken together, these data suggest that undirected singing behavior by non-breeding starlings is modulated by effects of DHEA on dopaminergic neurotransmission. More broadly, these data expand the social behavior functions of DHEA beyond territorial aggression to include undirected, affiliative social communication.

Unraveling the Role of Thyroid Hormones in Seasonal Neuroplasticity in European Starlings (Sturnus vulgaris).

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic–pituitary–gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.

Immunolabeling Provides Evidence for Subregions in the Songbird Nucleus Accumbens and Suggests a Context-Dependent Role in Song in Male European Starlings (Sturnus vulgaris)

Birdsong is well known for its role in mate attraction during the breeding season. However, many birds, including European starlings (Sturnus vulgaris), also sing outside the breeding season as part of large flocks. Song in a breeding context can be extrinsically rewarded by mate attraction; however, song in nonbreeding flocks, referred to here as gregarious song, results in no obvious extrinsic reward and is proposed to be intrinsically rewarded. The nucleus accumbens (NAC) is a brain region well known to mediate reward and motivation, which suggests it is an ideal candidate to regulate reward associated with gregarious song. The goal of this study was to provide new histochemical information on the songbird NAC and its subregions (rostral pole, core, and shell) and to begin to determine subregion-specific contributions to gregarious song in male starlings. We examined immunolabeling for tyrosine hydroxylase (TH), neurotensin, and enkephalin (ENK) in the NAC. We then examined the extent to which gregarious and sexually motivated song differentially correlated with immunolabeling for the immediate early genes FOS and ZENK in each subdivision of the NAC. We found that TH and ENK labeling within subregions of the starling NAC was generally similar to patterns seen in the core and shell of NACs in mammals and birds. Additionally, we found that gregarious song, but not sexually motivated song, positively correlated with FOS in all NAC subregions. Our observations provide further evidence for distinct subregions within the songbird NAC and suggest the NAC may play an important role in regulating gregarious song in songbirds.

Co-localization of mu-opioid and dopamine D1 receptors in the medial preoptic area and bed nucleus of the stria terminalis across seasonal states in male European starlings

In seasonally breeding animals, changes in photoperiod and sex-steroid hormones may modify sexual behavior in part by altering the activity of neuromodulators, including opioids and dopamine. In rats and birds, activation of mu-opioid receptors (MOR) and dopamine D1 receptors in the medial preoptic area (mPOA) often have opposing effects on sexual behavior, yet mechanisms by which the mPOA integrates these opposing effects to modulate behavior remain unknown. Here, we used male European starlings (Sturnus vulgaris) to provide insight into the hypothesis that MOR and D1 receptors modify sexual behavior seasonally by altering activity in the same neurons in the mPOA. To do this, using fluorescent immunohistochemistry, we examined the extent to which MOR and D1 receptors co-localize in mPOA neurons and the degree to which photoperiod and the sex-steroid hormone testosterone alter co-localization. We found that MOR and D1 receptors co-localize throughout the mPOA and the bed nucleus of the stria terminalis, a region also implicated in the control of sexual behavior. Numbers of single and co-labeled MOR and D1 receptor labeled cells were higher in the rostral mPOA in photosensitive males (a condition observed just prior to the breeding season) compared to photosensitive males treated with testosterone (breeding season condition). In the caudal mPOA co-localization of MOR and D1 receptors was highest in photosensitive males compared to photorefractory males (a post-breeding season condition). Seasonal shifts in the degree to which neurons in the mPOA integrate signaling from opioids and dopamine may underlie seasonal changes in the production of sexual behavior.

Environmental cues and dietary antioxidants affect breeding behavior and testosterone of male European starlings (Sturnus vulgaris)

Environmental cues, such as photoperiod, regulate the timing of major life-history events like breeding through direct neuroendocrine control. Less known is how supplementary environmental cues (e.g., nest sites, food availability) interact to influence key hormones and behaviors involved in reproduction, specifically in migratory species with gonadal recrudescence largely occurring at breeding sites. We investigated the behavioral and physiological responses of male European starlings to the sequential addition of nest boxes and nesting material, green herbs, and female conspecifics and how these responses depend on the availability of certain antioxidants (anthocyanins) in the diet. As expected, cloacal protuberance volume and plasma testosterone of males generally increased with photoperiod. More notably, testosterone levels peaked in males fed the high antioxidant diet when both nest box and herbal cues were present, while males fed the low antioxidant diet showed no or only a muted testosterone response to the sequential addition of these environmental cues; thus our results are in agreement with the oxidation handicap hypothesis. Males fed the high antioxidant diet maintained a constant frequency of breeding behaviors over time, whereas those fed the low antioxidant diet decreased breeding behaviors as environmental cues were sequentially added. Overall, sequential addition of the environmental cues modulated physiological and behavioral measures of reproductive condition, and dietary antioxidants were shown to be a key factor in affecting the degree of response to each of these cues. Our results highlight the importance of supplementary environmental cues and key resources such as dietary antioxidants in enhancing breeding condition of males, which conceivably aid in attraction of high quality females and reproductive success.

Co-localization patterns of neurotensin receptor 1 and tyrosine hydroxylase in brain regions involved in motivation and social behavior in male European starlings

Animals communicate in distinct social contexts to convey information specific to those contexts, such as sexual or agonistic motivation. In seasonally-breeding male songbirds, seasonal changes in day length and increases in testosterone stimulate sexually-motivated song directed at females for courtship and reproduction. Dopamine and testosterone may act in the same brain regions to stimulate sexually-motivated singing. The neuropeptide neurotensin, acting at the neurotensin receptor 1 (NTR1), can strongly influence dopamine transmission. The goal of this study was to gain insight into the degree to which seasonal changes in physiology modify interactions between neurotensin and dopamine to adjust context-appropriate communication. Male European starlings were examined in physiological conditions that stimulate season-typical forms of communication: late summer/early fall non-breeding condition (low testosterone; birds sing infrequently), late fall non-breeding condition (low testosterone; birds produce non-sexually motivated song), and spring breeding condition (high testosterone; males produce sexually-motivated song). Double fluorescent immunolabeling was performed to detect co-localization patterns between tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine synthesis) and NTR1 in brain regions implicated in motivation and song production (the ventral tegmental area, medial preoptic nucleus, periaqueductal gray, and lateral septum). Co-localization between TH and NTR1 was present in the ventral tegmental area for all physiological conditions, and the number of co-localized cells did not differ across conditions. Immunolabeling for TH and NTR1 was also present in the other examined regions, although no co-localization was seen. These results support the hypothesis that interactions between NTR1 and dopamine in the ventral tegmental area may modulate vocalizations, but suggest that testosterone- or photoperiod-induced changes in NTR1/TH co-localization do not underlie seasonally-appropriate adjustment of communication.

Topography and Lateralized Effect of Acute Aromatase Inhibition on Auditory Processing in a Seasonal Songbird.

It is increasingly recognized that brain-derived estrogens (neuroestrogens) can regulate brain physiology and behavior much faster than what was previously known from the transcriptional action of estrogens on nuclear receptors. One of the best examples of such neuromodulation by neuroestrogens concerns the acute regulation of sensory coding by the auditory cortex as demonstrated by electrophysiological studies of selected neurons in zebra finches. Yet, the spatial extent of such modulation by neuroestrogens is not known. Using functional magnetic resonance imaging, we demonstrate here that acute estrogen depletion alters within minutes auditory processing in male European starlings. These effects are confined to very specific but large areas of the auditory cortex. They are also specifically lateralized to the left hemisphere. Interestingly, the modulation of auditory responses by estrogens was much larger (both in amplitude and in topography) in March than in December or May/June. This effect was presumably independent from changes in circulating testosterone concentrations since levels of the steroid were controlled by subcutaneous implants, thus suggesting actions related to other aspects of the seasonal cycle or photoperiodic manipulations. Finally, we also show that estrogen production specifically modulates selectivity for behaviorally relevant vocalizations in a specific part of the caudomedial nidopallium. These findings confirm and extend previous conclusions that had been obtained by electrophysiological techniques. This approach provides a new very powerful tool to investigate auditory responsiveness in songbirds and its fast modulation by sex steroids.SIGNIFICANCE STATEMENT Neuroestrogens can acutely modulate sensory processing in a manner similar to neuromodulators. We report that acute estrogen depletion rapidly disrupts auditory processing in large areas of the male starling brain. Effects were larger in March than in December or May/June, lateralized to the left hemisphere and specific to behaviorally relevant stimuli. These findings confirm and extend previous data that identified an acute regulation of auditory neurons in zebra finches by (1) delineating the extent of the brain region affected, (2) confirming its lateralization, and (3) demonstrating that a large part of the auditory brain regions are acutely affected by estrogens. These findings provide a very powerful tool to investigate auditory responsiveness in songbirds and its fast modulation by sex steroids.

Experimental inhibition of a key cellular antioxidant affects vocal communication

Summary There is substantial interest of evolutionary ecologists in the proximate mechanisms that modulate vocal communication. In recent times, there has been growing interest in the role of oxidative stress as a mediator of avian song expression. Here, we tested whether the experimental inhibition of the synthesis of a key cellular antioxidant (glutathione) reduces song rate metrics of male European starlings (Sturnus vulgaris). We measured the effect of our treatment on total song rate and on its two components, undirected and nest‐box‐oriented song, outside the breeding season. Treated males that did not own a nest‐box (subordinate males likely to be of lower quality) suffered increased oxidative stress relative to untreated males, while treated males that owned a nest‐box (dominant males likely to be of higher quality) did not. Treated non‐owners also reduced their undirected song rate, whereas treated nest‐box owners did not suffer any reduction in song rate. Our results revealed that inhibition of a key cellular antioxidant results in decreased vocal communication in a social vertebrate, and that this effect is dependent on its social status (nest‐box owner vs. non‐owner). This work provides support for the hypothesis that acoustic signals may honestly convey information about the individual oxidative status and capacity to regulate the oxidative balance. Our findings raise the possibility of hitherto unexplored impacts of oxidative stress on fitness traits in social species. A lay summary is available for this article.

Song-associated reward correlates with endocannabinoid-related gene expression in male European starlings (Sturnus vulgaris)

Vocal communication is required for successful social interactions in numerous species. During the breeding season, songbirds produce songs that are reinforced by behavioral consequences (e.g., copulation). However, some songbirds also produce songs not obviously directed at other individuals. The consequences maintaining or reinforcing these songs are less obvious and the neural mechanisms associated with undirected communication are not well-understood. Previous studies indicate that undirected singing is intrinsically rewarding and mediated by opioid or dopaminergic systems; however, endocannabinoids are also involved in regulating reward and singing behavior. We used a conditioned place preference paradigm to examine song-associated reward in European starlings and quantitative real-time PCR to measure expression of endocannabinoid-related neural markers (CB1, FABP7, FABP5, FAAH, DAGLα), in brain regions involved in social behavior, reward and motivation (ventral tegmental area [VTA], periaqueductal gray [PAG], and medial preoptic nucleus [POM]), and a song control region (Area X). Our results indicate that starlings producing high rates of song developed a conditioned place preference, suggesting that undirected song is associated with a positive affective state. We found a significant positive relationship between song-associated reward and CB1 receptors in VTA and a significant negative relationship between song-associated reward and CB1 in PAG. There was a significant positive relationship between reward and the cannabinoid transporter FABP7 in POM and a significant negative relationship between reward and FABP7 in PAG. In Area X, FABP5 and DAGLα correlated positively with singing. These results suggest a role for endocannabinoid signaling in vocal production and reward associated with undirected communication.

Prodynorphin and kappa opioid receptor mRNA expression in the brain relates to social status and behavior in male European starlings

Numerous animal species display behavioral changes in response to changes in social status or territory possession. For example, in male European starlings only males that acquire nesting sites display high rates of sexual and agonistic behavior. Past studies show that mu and delta opioid receptors regulate behaviors associated with social ascension or defeat. Opioids also act at kappa receptors, with dynorphin binding with the highest affinity; however, the role of these opioids in social behavior has not been well studied. We observed flocks of male starlings during the breeding season and ran quantitative real-time polymerase chain reaction (qPCR) to measure expression of kappa opioid receptors (OPRK1) and prodynorphin (PDYN) in brain regions involved in social behavior and motivation (ventral tegmental area [VTA], medial preoptic nucleus [mPOA]) and vocal behavior (Area X). Males with nesting territories displayed more sexual/agonistic behavior than males without nesting territories. They also had lower OPRK1 expression in VTA and mPOA. OPRK1 expression in VTA correlated negatively with sexual/agonistic behaviors, consistent with past studies showing kappa receptors in VTA to inhibit sociosexual behaviors. PDYN in mPOA correlated negatively with a measure of nesting behavior that may also reflect sexual motivation. PDYN in Area X related positively to song. Distinct patterns of OPRK1 and PDYN expression in VTA, mPOA, and Area X related to gonad volume, suggesting that breeding condition may modify (or be modified by) OPRK1 and PDYN expression. Studies are now needed to further characterize the role of OPRK1 and PDYN in status-appropriate social behaviors.

Contributions of testosterone and territory ownership to sexually-motivated behaviors and mRNA expression in the medial preoptic area of male European starlings

Animals integrate social information with their internal endocrine state to control the timing of behavior, but how these signals are integrated in the brain is not understood. The medial preoptic area (mPOA) may play an integrative role in the control of courtship behavior, as it receives projections from multiple sensory systems, and is central to the hormonal control of courtship behavior across vertebrates. Additionally, data from many species implicate opioid and dopaminergic systems in the mPOA in the control of male courtship behavior. We used European starlings to test the hypothesis that testosterone (T) and social status (in the form of territory possession) interact to control the timing of courtship behavior by modulating steroid hormone-, opioid- and dopaminergic-related gene expression in the mPOA. We found that only males given both T and a nesting territory produced high rates of courtship behavior in response to a female. T treatment altered patterns of gene expression in the mPOA by increasing androgen receptor, aromatase, mu-opioid receptor and preproenkephalin mRNA and decreasing tyrosine hydroxylase mRNA expression. Territory possession did not alter mRNA expression in the mPOA, despite the finding that only birds with both T and a nesting territory produced courtship behavior. We propose that T prepares the mPOA to respond to the presence of a female with high rates of courtship song by altering gene expression, but that activity in the mPOA is under a continuous (i.e. tonic) inhibition until a male starling obtains a nesting territory.

Song in an Affiliative Context Relates to the Neural Expression of Dopamine- and Neurotensin-Related Genes in Male European Starlings

Some animals, including songbirds, vocalize at high rates when alone or in large groups. In songbirds, vocal behavior in these contexts is important for song learning and group cohesion. It is not obviously targeted at any particular individual and is described as ‘undirected'. Studies suggest a role for dopamine (DA) in undirected song. The neuropeptide neurotensin (NT) can enhance dopaminergic signaling upon binding to the NT receptor 1 (NTR1) and is found in regions where DA can influence song, including the ventral tegmental area (VTA), septum, and the song control nucleus Area X. To begin to test the hypothesis that NT and DA in these regions interact to influence undirected song, we used quantitative real-time PCR to relate undirected singing to mRNA expression of NT, NTR1, tyrosine hydroxylase (TH; a synthetic enzyme for DA) and D1 and D2 receptors in male European starlings. TH and NT expression in VTA, and NT and D1 expression in Area X, positively correlated with song. NT markers also correlated positively with DA markers in VTA. Given the role of VTA projections to Area X in song learning, these results suggest that interactions between NT and DA in these regions may contribute to vocal learning. In septum, NTR1 expression positively correlated with song and NT and DA markers were correlated, suggesting that NT in this region may influence dopaminergic transmission to facilitate undirected vocalizations. Overall, these findings implicate interactions between NT and DA in affiliative communication.

Neural endocannabinoid CB1 receptor expression, social status, and behavior in male European starlings

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.

A novel statistical method for behaviour sequence analysis and its application to birdsong

Complex vocal signals, such as birdsong, contain acoustic elements that differ in both order and duration. These elements may convey socially relevant meaning, both independently and through their interactions, yet statistical methods that combine order and duration data to extract meaning have not, to our knowledge, been fully developed. Here we design novel semi-Markov methods, Bayesian estimation and classification trees to extract order and duration information from behavioural sequences and apply these methods to songs produced by male European starlings, Sturnus vulgaris, in two social contexts in which the function of song differs: a spring (breeding) and autumn (nonbreeding) context. Additionally, previous data indicate that damage to the medial preoptic nucleus (POM), a brain area known to regulate male sexually motivated behaviour, affects structural aspects of starling song such that males in a sexually relevant context (i.e. spring) sing shorter songs than appropriate for this context. We further test the utility of our statistical approach by comparing attributes of song structure in POM-lesioned males to song produced by control spring and autumn males. Spring and autumn songs were statistically separable based on the duration and order of phrase types. Males produced more structurally complex aspects of song in spring than in autumn. Spring song was also longer and more stereotyped than autumn song, both attributes used by females to select mates. Songs produced by POM-lesioned males in some cases fell between measures of spring and autumn songs but differed most from songs produced by autumn males. Overall, these statistical methods can effectively extract biologically meaningful information contained in many behavioural sequences given sufficient sample sizes and replication numbers.

Neurotensin neural mRNA expression correlates with vocal communication and other highly-motivated social behaviors in male European starlings

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 animal groups, 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 sexually-motivated 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.

Differential relationships between D1 and D2 dopamine receptor expression in the medial preoptic nucleus and sexually-motivated song in male European starlings (Sturnus vulgaris)

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.

Birds receiving extra carotenoids keep singing during the sickness phase induced by inflammation

Life history theory predicts that individuals have to trade-off resources between diverse energy-demanding activities, such as mounting an immune response and performing advertisement behaviour. The availability of immunomodulatory micronutrients can affect this trade-off. Carotenoids can upregulate both the humoral and cell-mediated immune response, but little is known about their effect on behavioural traits during the sickness phase induced by a common inflammation. To investigate whether dietary carotenoids can mitigate the severity of the sickness syndrome and promote fitness-related traits, we studied how the song rate of captive male European starlings (Sturnus vulgaris, Linnaeus 1758) receiving dietary carotenoids and coping with a challenge (lipopolysaccharide, LPS) mimicking a bacterial infection varied during the sickness phase and the subsequent recovery phase. We found that birds not provisioned with carotenoids and injected with LPS sang less than control birds during the sickness phase, but not during the recovery phase. Conversely, birds provided access to a carotenoid-enriched diet never decreased their song rate. Our results show that immune-challenged birds have to trade-off between mounting an immune response and advertising only when their access to dietary carotenoids is limited. No differences in song rate were observed between treatments during the recovery phase. Our study is the first to investigate the role of dietary carotenoids on a behavioural syndrome-like sickness and to show that providing a carotenoid-rich diet can alleviate the social costs associated with the occurrence of an inflammation.