The molecular architecture of a complex social behavior: gregarious song.

The medial preoptic area (mPOA) regulates the probability and intensity of singing behavior in birds. Polzin and colleagues examined the molecular changes in the mPOA that were associated with gregarious song in European starlings (Sturnus vulgaris). High-throughput transcriptome analyses identified glutamate and dopamine pathways were highly enriched with gregarious song.

Distinct patterns of gene expression in the medial preoptic area are related to gregarious singing behavior in European starlings (Sturnus vulgaris).

BACKGROUND: Song performed in flocks by European starlings (Sturnus vulgaris), referred to here as gregarious song, is a non-sexual, social behavior performed by adult birds. Gregarious song is thought to be an intrinsically reinforced behavior facilitated by a low-stress, positive affective state that increases social cohesion within a flock. The medial preoptic area (mPOA) is a region known to have a role in the production of gregarious song. However, the neurochemical systems that potentially act within this region to regulate song remain largely unexplored. In this study, we used RNA sequencing to characterize patterns of gene expression in the mPOA of male and female starlings singing gregarious song to identify possibly novel neurotransmitter, neuromodulator, and hormonal pathways that may be involved in the production of gregarious song. RESULTS: Differential gene expression analysis and rank rank hypergeometric analysis indicated that dopaminergic, cholinergic, and GABAergic systems were associated with the production of gregarious song, with multiple receptor genes (e.g., DRD2, DRD5, CHRM4, GABRD) upregulated in the mPOA of starlings who sang at high rates. Additionally, co-expression network analyses identified co-expressing gene clusters of glutamate signaling-related genes associated with song. One of these clusters contained five glutamate receptor genes and two glutamate scaffolding genes and was significantly enriched for genetic pathways involved in neurodevelopmental disorders associated with social deficits in humans. Two of these genes, GRIN1 and SHANK2, were positively correlated with performance of gregarious song. CONCLUSIONS: This work provides new insights into the role of the mPOA in non-sexual, gregarious song in starlings and highlights candidate genes that may play a role in gregarious social interactions across vertebrates. The provided data will also allow other researchers to compare across species to identify conserved systems that regulate social behavior.

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).

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

Structural insights into the chromodomain of Oxpecker in complex with histone H3 lysine 9 trimethylation reveal a transposon silencing mechanism by heterodimerization.

Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. In this study, we solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, we found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD.

Flight training and dietary antioxidants have mixed effects on the oxidative status of multiple tissues in a female migratory songbird.

Birds, like other vertebrates, rely on a robust antioxidant system to protect themselves against oxidative imbalance caused by energy-intensive activities such as flying. Such oxidative challenges may be especially acute for females during spring migration, as they must pay the oxidative costs of flight while preparing for reproduction; however, little previous work has examined how the antioxidant system of female spring migrants responds to dietary antioxidants and the oxidative challenges of regular flying. We fed two diets to female European starlings, one supplemented with a dietary antioxidant and one without, and then flew them daily in a windtunnel for 2 weeks during the autumn and spring migration periods. We measured the activity of enzymatic antioxidants (glutathione peroxidase, superoxide dismutase and catalase), non-enzymatic antioxidant capacity (ORAC) and markers of oxidative damage (protein carbonyls and lipid hydroperoxides) in four tissues: pectoralis, leg muscle, liver and heart. Dietary antioxidants affected enzymatic antioxidant activity and lipid damage in the heart, non-enzymatic antioxidant capacity in the pectoralis, and protein damage in leg muscle. In general, birds not fed the antioxidant supplement appeared to incur increased oxidative damage while upregulating non-enzymatic and enzymatic antioxidant activity, though these effects were strongly tissue specific. We also found trends for diet×training interactions for enzymatic antioxidant activity in the heart and leg muscle. Flight training may condition the antioxidant system of females to dynamically respond to oxidative challenges, and females during spring migration may shift antioxidant allocation to reduce oxidative damage.

µ-Opioid Receptor Stimulation in the Nucleus Accumbens Increases Vocal-Social Interactions in Flocking European Starlings, Sturnus Vulgaris.

Social connections in gregarious species are vital for safety and survival. For these reasons, many bird species form large flocks outside the breeding season. It has been proposed that such large social groups may be maintained via reward induced by positive interactions with conspecifics and via the reduction of a negative affective state caused by social separation. Moreover, within a flock optimal social spacing between conspecifics is important, indicating that individuals may optimize spacing to be close but not too close to conspecifics. The µ-opioid receptors (MORs) in the nucleus accumbens (NAc) are well known for their role in both reward and the reduction of negative affective states, suggesting that MOR stimulation in NAc may play a critical role in flock cohesion. To begin to test this hypothesis, social and nonsocial behaviors were examined in male and female European starlings (Sturnus vulgaris) in nonbreeding flocks after intra-NAc infusion of saline and three doses of the selective MOR agonist d-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO). DAMGO in NAc dose-dependently increased singing behavior and facilitated social approaches while at the same time promoting displacements potentially used to maintain social spacing. These findings support the hypothesis that MORs in NAc promote social interactions important for group cohesion in nonsexual contexts and suggest the possibility that MORs in the NAc play a role in optimizing the pull of joining a flock with the push of potential agonistic encounters.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank-Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank-Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

The energy savings-oxidative cost trade-off for migratory birds during endurance flight.

Elite human and animal athletes must acquire the fuels necessary for extreme feats, but also contend with the oxidative damage associated with peak metabolic performance. Here, we show that a migratory bird with fuel stores composed of more omega-6 polyunsaturated fats (PUFA) expended 11% less energy during long-duration (6 hr) flights with no change in oxidative costs; however, this short-term energy savings came at the long-term cost of higher oxidative damage in the omega-6 PUFA-fed birds. Given that fatty acids are primary fuels, key signaling molecules, the building blocks of cell membranes, and that oxidative damage has long-term consequences for health and ageing, the energy savings-oxidative cost trade-off demonstrated here may be fundamentally important for a wide diversity of organisms on earth.

Flight training in a migratory bird drives metabolic gene expression in the flight muscle but not liver, and dietary fat quality influences select genes.

Training and diet are hypothesized to directly stimulate key molecular pathways that mediate animal performance, and flight training, dietary fats, and dietary antioxidants are likely important in modulating molecular metabolism in migratory birds. This study experimentally investigated how long-distance flight training, as well as diet composition, affected the expression of key metabolic genes in the pectoralis muscle and the liver of European starlings (Sturnus vulgaris, n = 95). Starlings were fed diets composed of either a high or low polyunsaturated fatty acid (PUFA; 18:2n-6) and supplemented with or without a water-soluble antioxidant, and one-half of these birds were flight trained in a wind-tunnel while the rest were untrained. We measured the expression of 7 (liver) or 10 (pectoralis) key metabolic genes in flight-trained and untrained birds. Fifty percent of genes involved in mitochondrial metabolism and fat utilization were upregulated by flight training in the pectoralis (P < 0.05), whereas flight training increased the expression of only one gene responsible for fatty acid hydrolysis [lipoprotein lipase (LPL)] in the liver (P = 0.04). Dietary PUFA influenced the gene expression of LPL and fat transporter fatty acid translocase (CD36) in the pectoralis and one metabolic transcription factor [peroxisome proliferator-activated receptor (PPAR)-α (PPARα)] in the liver, whereas dietary antioxidants had no effect on the metabolic genes measured in this study. Flight training initiated a simpler causal network between PPARγ coactivators, PPARs, and metabolic genes involved in mitochondrial metabolism and fat storage in the pectoralis. Molecular metabolism is modulated by flight training and dietary fat quality in a migratory songbird, indicating that these environmental factors will affect the migratory performance of birds in the wild.

Meat quality and lipid fatty acid profile from wild thrush (Turdus philomelos), woodcock (Scolopax rusticola) and starling (Sturnus vulgaris): a preliminary comparative study.

BACKGROUND: The present study aimed to evaluate the nutritional proximate composition, some qualitative traits and fatty acid profile of meat from wild thrush, woodcock and starling hunted in Southern Italy in 2017 and 2018. METHODS: Nutritive composition and physical traits of meat and lipid fatty acid profile were evaluated in breast muscle (Pectoralis major) of gamebirds. RESULTS: From findings, the meat pH was significantly (P < 0.001) higher in starling when compared to the other two species. Thrush meat was significantly (P = 0.002) darker and had higher redness (P < 0.001) and yellowness (P = 0.004) in comparison to starling and woodcock. Thrush breast muscle showed the highest (P < 0.001) level of lipids and lowest (P < 0.001) protein content. Meat from thrush showed the best lipid fatty acid profile based on the higher (P < 0.001) monounsaturated fatty acids (MUFA) and lower (P < 0.001) saturated fatty acids (SFA) concentrations. Starling breast muscle reported the highest (P = 0.002) polyunsaturated fatty acids (PUFA) level compared to both thrush and woodcock, whereas no differences were detected on total n-3. The ratio n-6/n-3 was higher (P = 0.001) in starling muscle. Thrush breast muscle had the lowest (P < 0.001) atherogenic and thrombogenic indices compared to the other gamebirds. CONCLUSIONS: The findings indicated that meat from the three investigated gamebirds species may represent a healthily lipid food source for human consumption in relation to the prevention of cardiovascular diseases.

Characterizing the timing of yolk testosterone metabolism and the effects of etiocholanolone on development in avian eggs.

Maternal transfer of steroids to eggs can elicit permanent effects on offspring phenotype. Although testosterone was thought to be a key mediator of maternal effects in birds, we now know that vertebrate embryos actively regulate their exposure to maternal testosterone through steroid metabolism, suggesting testosterone metabolites, not testosterone, may elicit the observed phenotypic effects. To address the role steroid metabolism plays in mediating yolk testosterone effects, we used European starling (Sturnus vulgaris) eggs to characterize the timing of testosterone metabolism and determine whether etiocholanolone, a prominent metabolite of testosterone in avian embryos, is capable of affecting early embryonic development. Tritiated testosterone was injected into freshly laid eggs to characterize steroid movement and metabolism during early development. Varying levels of etiocholanolone were also injected into eggs, with incubation for either 3 or 5 days, to test whether etiocholanolone influences the early growth of embryonic tissues. The conversion of testosterone to etiocholanolone was initiated within 12 h of injection, but the increase in etiocholanolone was transient, indicating that etiocholanolone is also subject to metabolism, and that exposure to maternal etiocholanolone is limited to a short period during early development. Exogenous etiocholanolone manipulation had no significant effect on the growth rate of the embryos or extra-embryonic membranes early in development. Thus, the conversion of testosterone to etiocholanolone may be an inactivation pathway that buffers the embryo from maternal steroids, with any effects of yolk testosterone resulting from testosterone that escapes metabolism; alternatively, etiocholanolone may influence processes other than growth or take additional time to manifest.

Bioaccumulation and biomagnification of PBDEs in a terrestrial food chain at an urban landfill.

Biota samples from the Vancouver municipal landfill located in Delta, BC, Canada, have some of the highest polybrominated diphenyl ether (PBDE) levels reported from North America. We followed a population of European starlings (Sturnus vulgaris) breeding in a remediated area in the landfill to identify exposure routes and bioaccumulation of PBDEs in a simple terrestrial food chain. This population was compared to a reference farm site located 40 km east in Glen Valley. We analyzed samples of European starling eggs and nestling livers as well as invertebrate prey species consumed by starlings for PBDE concentrations. We also collected soil samples from starling foraging areas. All samples from the Delta landfill had higher PBDE congener concentrations compared to the Glen Valley reference site and were dominated by BDE-99 and BDE-47. Stable nitrogen (δ N15) and carbon (δ C13) isotope analysis of starling blood samples and provisioned invertebrates revealed that stable δC13 signatures differed between the sites indicating that the diet of starlings in the Delta landfill included a component of human refuse. Biota-soil accumulation factors (BSAFs) > 1 demonstrated that PBDEs were bioaccumulating in soil invertebrates, particularly earthworms, which were readily accessible to foraging starlings in the landfill. Biomagnification factors (BMFs) calculated from foraged food items and starling egg and liver samples were >1, indicating that a diet of soil invertebrates and refuse contributed substantially to the PBDE exposure of local starlings.

A Tissue Comparison of DNA Methylation of the Glucocorticoid Receptor Gene (Nr3c1) in European Starlings.

Negative feedback of the vertebrate stress response via the hypothalamic-pituitary-adrenal (HPA) axis is regulated by glucocorticoid receptors in the brain. Epigenetic modification of the glucocorticoid receptor gene (Nr3c1), including DNA methylation of the promoter region, can influence expression of these receptors, impacting behavior, physiology, and fitness. However, we still know little about the long-term effects of these modifications on fitness. To better understand these fitness effects, we must first develop a non-lethal method to assess DNA methylation in the brain that allows for multiple measurements throughout an organism's lifetime. In this study, we aimed to determine if blood is a viable biomarker for Nr3c1 DNA methylation in two brain regions (hippocampus and hypothalamus) in adult European starlings (Sturnus vulgaris). We found that DNA methylation of CpG sites in the complete Nr3c1 putative promoter varied among tissue types and was lowest in blood. Although we identified a similar cluster of correlated Nr3c1 putative promoter CpG sites within each tissue, this cluster did not show any correlation in DNA methylation among tissues. Additional studies should consider the role of the developmental environment in producing epigenetic modifications in different tissues.

Conditioned place preferences induced by hearing song outside the breeding season relate to neural dopamine D1 and cannabinoid CB1 receptor gene expression in female European starlings (Sturnus vulgaris).

The affective state induced by sensory stimuli changes to adaptively modify behaviors that are critical for survival and reproduction. In European starlings, during the spring breeding season, male courtship song is rewarding to females, but only to those that possess resources that are necessary for reproduction (i.e., nesting sites). In fall, starling song is non-sexual and proposed to maintain flocks. This suggests that in fall it may be adaptive for females to be rewarded by fall rather than spring, courtship song. We used a conditioned place preference (CPP) test to evaluate song-induced affective state in fall condition females and quantitative real-time PCR to measure expression of genes that modulate affective state (CB1 endocannabinoid and D1 dopamine receptors) in brain regions that were previously implicated in song-induced reward (i.e., the medial preoptic nucleus (mPOA) and ventromedial hypothalamus (VMH)). Fall condition females developed an aversion to a place that had been paired with playback of both male fall and courtship song, indicating that in general male song induces a negative affective state outside the breeding season. Song-induced aversion was stronger in birds conditioned towards an initial place preference. For mPOA, CB1 receptor expression correlated positively with fall and spring song-induced CPP. D1 receptor expression correlated negatively with fall (but not spring) song-induced CPP, and the ratio of CB1 to D1 receptor expression correlated positively with fall (but not spring) song-induced CPP. These correlations suggest that interactions between D1 and CB1 receptors in mPOA may play a role in modifying affective responses to song.

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.

Nest box exploration may stimulate breeding physiology and alter mRNA expression in the medial preoptic area of female European starlings.

Environmental resources are proposed to fine-tune the timing of breeding, yet how they may do so remains unclear. In female European starlings (Sturnus vulgaris), nest cavities are limited resources that are necessary for breeding. Females that explore nest cavities, compared with those that do not, readily perform sexually motivated behaviors. We assigned female starlings to aviaries with: (1) no nest boxes, (2) nest boxes, or (3) nest boxes, plants, flowing water, insects and berries to test the hypothesis that environmental resources alter neural systems to stimulate mating behavior. Compared with other females, females that were housed with and explored nest boxes had higher estradiol, higher preproenkephalin (PENK) mRNA and lower levels of D1 and D2 dopamine receptor mRNA in the medial preoptic area (mPOA); a region in which opioids and dopamine modify female sexual behaviors and sexual motivation. Additionally, in the mPOA, PENK and tyrosine hydroxylase mRNA positively predicted, whereas estrogen receptor beta mRNA negatively predicted, nest box exploration. In the ventromedial hypothalamus (a region in which estradiol acts to stimulate sexual behavior), estrogen receptor alpha mRNA was highest in females that had access to but did not explore nest cavities. It is likely that seasonal increases in estradiol modify mRNA in the mPOA to facilitate nest cavity exploration. It is also possible that nest cavity exploration further alters gene expression in the mPOA, functioning to coordinate mating with resource availability. Thus, nest cavity exploration may be a form of self-stimulation that alters neural systems to fine-tune sexual behavior.

Rapid depuration of mercury in songbirds accelerated by feather molt.

Mercury (Hg) is a ubiquitous environmental contaminant that affects avian reproduction and condition, in both aquatic and terrestrial species. Because Hg binds strongly to the keratin of growing feathers, molt is an important avenue for Hg elimination. We investigated the rate of depuration of Hg from songbird blood and organs (brain, kidney, liver, muscle) as a function of molt. We exposed 2 species of captive songbirds, European starling (Sturnus vulgaris) and zebra finch (Taeniopygia guttata), to environmentally relevant dietary Hg (1.2-1.5 mg/kg wet wt) for extended periods of time and then allowed them to depurate on a Hg-free diet for 20 wk. Depuration occurred either while birds were regrowing new feathers or when there was little to no molt occurring. Both species quickly eliminated Hg from blood and reduced Hg concentrations in other tissues by more than 90% over 20 wk. The rate of depuration was more rapid than previously reported in the literature, but depuration in small-bodied songbirds has rarely been studied. Molting birds depurated Hg from their blood more rapidly than nonmolting birds. The effect of molt on Hg retention in other tissues was more difficult to resolve, because by the time we sampled organ tissue, depuration was nearly complete. Our results confirm that molting expedites depuration and raises mechanistic questions about how songbirds depurate Hg stored in their tissues. Environ Toxicol Chem 2017;36:3120-3126. © 2017 SETAC.

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.

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed-ended and open-ended learning.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.