Expression of FoxP2 in the basal ganglia regulates vocal motor sequences in the adult songbird.

Disruption of the transcription factor FoxP2, which is enriched in the basal ganglia, impairs vocal development in humans and songbirds. The basal ganglia are important for the selection and sequencing of motor actions, but the circuit mechanisms governing accurate sequencing of learned vocalizations are unknown. Here, we show that expression of FoxP2 in the basal ganglia is vital for the fluent initiation and termination of birdsong, as well as the maintenance of song syllable sequencing in adulthood. Knockdown of FoxP2 imbalances dopamine receptor expression across striatal direct-like and indirect-like pathways, suggesting a role of dopaminergic signaling in regulating vocal motor sequencing. Confirming this prediction, we show that phasic dopamine activation, and not inhibition, during singing drives repetition of song syllables, thus also impairing fluent initiation and termination of birdsong. These findings demonstrate discrete circuit origins for the dysfluent repetition of vocal elements in songbirds, with implications for speech disorders.

Cellular transcriptomics reveals evolutionary identities of songbird vocal circuits.

Birds display advanced behaviors, including vocal learning and problem-solving, yet lack a layered neocortex, a structure associated with complex behavior in mammals. To determine whether these behavioral similarities result from shared or distinct neural circuits, we used single-cell RNA sequencing to characterize the neuronal repertoire of the songbird song motor pathway. Glutamatergic vocal neurons had considerable transcriptional similarity to neocortical projection neurons; however, they displayed regulatory gene expression patterns more closely related to neurons in the ventral pallium. Moreover, while γ-aminobutyric acid-releasing neurons in this pathway appeared homologous to those in mammals and other amniotes, the most abundant avian class is largely absent in the neocortex. These data suggest that songbird vocal circuits and the mammalian neocortex have distinct developmental origins yet contain transcriptionally similar neurons.

In vivo imaging of D2 receptors and corticosteroids predict behavioural responses to captivity stress in a wild bird.

Individual physiological variation may underlie individual differences in behaviour in response to stressors. This study tested the hypothesis that individual variation in dopamine and corticosteroid physiology in wild house sparrows (Passer domesticus, n = 15) would significantly predict behaviour and weight loss in response to a long-term stressor, captivity. We found that individuals that coped better with captivity (fewer anxiety-related behaviours, more time spent feeding, higher body mass) had lower baseline and higher stress-induced corticosteroid titres at capture. Birds with higher striatal D2 receptor binding (examined using positron emission tomography (PET) with 11C-raclopride 24 h post-capture) spent more time feeding in captivity, but weighed less, than birds with lower D2 receptor binding. In the subset of individuals imaged a second time, D2 receptor binding decreased in captivity in moulting birds, and larger D2 decreases were associated with increased anxiety behaviours 2 and 4 weeks post-capture. This suggests changes in dopaminergic systems could be one physiological mechanism underlying negative behavioural effects of chronic stress. Non-invasive technologies like PET have the potential to transform our understanding of links between individual variation in physiology and behaviour and elucidate which neuroendocrine phenotypes predict stress resilience, a question with important implications for both humans and wildlife.

Beyond bulk: a review of single cell transcriptomics methodologies and applications.

Single-cell RNA sequencing (scRNA-seq) is a promising approach to study the transcriptomes of individual cells in the brain and the central nervous system (CNS). This technology acts as a bridge between neuroscience, computational biology, and systems biology, enabling an unbiased and novel understanding of the cellular composition of the brain and CNS. Gene expression at the single cell resolution is often noisy, sparse, and high-dimensional, creating challenges for computational analysis of such data. In this review, we overview fundamental sample preparation and data analysis processes of scRNA-seq and provide a comparative perspective for analyzing and visualizing these data.

Song practice as a rewarding form of play in songbirds.

In adult songbirds, the primary functions of song are mate attraction and territory defense; yet, many songbirds sing at high rates as juveniles and outside these primary contexts as adults. Singing outside primary contexts is critical for song learning and maintenance, and ultimately necessary for breeding success. However, this type of singing (i.e., song "practice") occurs even in the absence of immediate or obvious extrinsic reinforcement; that is, it does not attract mates or repel competitors. Here we review studies that support the hypothesis that song practice is stimulated and maintained by intrinsic reward mechanisms (i.e., that it is associated with a positive affective state). Additionally, we propose that song practice can be considered a rewarding form of play behavior similar to forms of play observed in multiple young animals as they practice sequences of motor events that are used later in primary adult reproductive contexts. This review highlights research suggesting at least partially overlapping roles for neural reward systems in birdsong and mammalian play and evidence that steroid hormones modify these systems to shift animals from periods of intrinsically rewarded motor exploration (i.e., singing in birds and play in mammals) to the use of similar motor patterns in primary reproductive contexts.

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.

Neurotensin and neurotensin receptor 1 mRNA expression in song-control regions changes during development in male zebra finches.

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine-related measures change throughout development in song-control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song-control regions of male zebra finches in four stages of the song learning process: pre-subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre-subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671-686, 2018.

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.

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.

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.

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.

Neurotensin immunolabeling relates to sexually-motivated song and other social behaviors in male European starlings (Sturnus vulgaris).

The brain regions involved in vocal communication are well described for some species, including songbirds, but less is known about the neural mechanisms underlying motivational aspects of communication. Mesolimbic dopaminergic projections from the ventral tegmental area (VTA) are central to mediating motivated behaviors. In songbirds, VTA provides dopaminergic innervation to brain regions associated with motivation and social behavior that are also involved in sexually-motivated song production. Neurotensin (NT) is a neuropeptide that strongly modulates dopamine activity, co-localizes with dopamine in VTA, and is found in regions where dopaminergic cells project from VTA. Yet, little is known about how NT contributes to vocal communication or other motivated behaviors. We examined the relationships between sexually-motivated song produced by male European starlings (Sturnus vulgaris) and NT immunolabeling in brain regions involved in social behavior and motivation. Additionally, we observed relationships between NT labeling, non-vocal courtship behaviors (another measure of sexual motivation), and agonistic behavior to begin to understand NT's role in socially-motivated behaviors. NT labeling in VTA, lateral septum, and bed nucleus of the stria terminalis correlated with sexually-motivated singing and non-vocal courtship behaviors. NT labeling in VTA, lateral septum, medial preoptic nucleus, and periaqueductal gray was associated with agonistic behavior. This study is the first to suggest NT's involvement in song, and one of the few to implicate NT in social behaviors more generally. Additionally, our results are consistent with the idea that distinct patterns of neuropeptide activity in brain areas involved in social behavior and motivation underlie differentially motivated behaviors.