Central prolactin receptor distribution and pSTAT5 activation patterns in breeding and non-breeding zebra finches (Taeniopygia guttata).

The hormone prolactin has many diverse functions across taxa such as osmoregulation, metabolism, and reproductive behavior. In ring doves, central prolactin action is important for parental care and feeding behavior. However, there is a considerable lack of information on the distribution of the prolactin receptor (PRLR) in the avian CNS to test the hypothesis that prolactin mediates these and other functions in other birds. In order to advance this research, we collected brains from breeding and non-breeding zebra finches to map the PRLR distribution using immunohistochemistry. We found PRLRs are distributed widely across the brain, both in hypothalamic sites known to regulate parental care and feeding, but also in many non-hypothalamic sites, including the tectofugal visual pathway, song system regions, reward associated areas, and pallium. This raises the possibility that prolactin has other functions throughout the brain that are not necessarily related to feeding or parental care. In addition, we also stained brains for pSTAT5, a transcription factor which is expressed when the PRLR is activated and is used as a marker for PRLR activity. We found several notable differences in pSTAT5 activity due to the breeding state of the animal, in both directions, further supporting the hypothesis that prolactin has many diverse functions in the brain both within and outside times of breeding. Together, this study represents the first essential step to inform the design of causative studies which manipulate PRLR-expressing cells to test their role in a wide variety of behaviors and other physiological functions.

Sexual and pairing partner preference in birds and other animals.

Research on hormonal involvement in animals' preferences for mating or pairing with same- or other-sex partners has been among the topics featured in Hormones and Behavior over the years. In several species of non-pair-forming mammals, there is good evidence that the early sex steroid environment has an organizational effect on later sexual partner preference. Research with zebra finches, a pair-forming species, shows a likely early estrogenic organizational hormone effect on pairing partner preference, an effect that can also interact with the early social environment to determine adult pairing preference. Experiments with two amphibian and fish species suggest that activational hormone effects (effects of the hormone milieu in adulthood) may regulate sexual partner preference. As a complement to the research on hormonal involvement, a growing body of theoretical and empirical research addresses the possible adaptive functions of the same-sex sexual and pairing behavior observed in many wild animals. Such advances have important implications for conceptualizing neuroendocrine mechanisms of partner preferences.

Do nonapeptides regulate parental care depending on experience in zebra finches?

Recent research suggests that the nonapeptide neurohormones regulate parental behaviors in a diverse array of vertebrates. However, it remains unclear how these neurohormones regulate parental care among birds, especially those which exhibit biparental care, or whether hormonal effects are contingent on a bird's previous experience as a parent. We measured the effects of nonapeptides on parental behaviors by peripherally injecting, over three treatment days, a short-acting nonapeptide receptor antagonist (OTA) or a saline control into breeding pairs of zebra finches (Taeniopygia guttata) that either did or did not have previous parental experience. We then compared how the duration of parental behaviors changed over the five days of observation (including one day before and two days after injections were administered). To compare treatment effects on parental outcomes, we also measured chick growth and mortality rates for each pair. There was a nearly significant interaction between treatment and experience for the amount of time birds spent in the nest, with time in the nest declining across the experiment inexperienced and experienced OTA birds. There was also a significant treatment by trial day interaction for nest guarding and a treatment by experience by trial day interaction for nest maintenance. Chicks reared by parents that received the OTA had significantly lower growth rates than chicks reared by control parents and, among experienced birds, higher mortality relative to control birds. Together, these results provide some support for the hypothesis that nonapeptides play a role in regulating parental outcomes and some parental behaviors in both experienced and inexperienced zebra finches.

Co-opting evo-devo concepts for new insights into mechanisms of behavioural diversity.

We propose that insights from the field of evolutionary developmental biology (or 'evo-devo') provide a framework for an integrated understanding of the origins of behavioural diversity and its underlying mechanisms. Towards that goal, in this Commentary, we frame key questions in behavioural evolution in terms of molecular, cellular and network-level properties with a focus on the nervous system. In this way, we highlight how mechanistic properties central to evo-devo analyses - such as weak linkage, versatility, exploratory mechanisms, criticality, degeneracy, redundancy and modularity - affect neural circuit function and hence the range of behavioural variation that can be filtered by selection. We outline why comparative studies of molecular and neural systems throughout ontogeny will provide novel insights into diversity in neural circuits and behaviour.

Lowering prolactin reduces post-hatch parental care in male and female zebra finches (Taeniopygia guttata).

Parental care is a widespread phenomenon observed in many diverse taxa. Neuroendocrine systems have long been thought to play an important role in stimulating the onset of parental behavior. In most birds with altricial young, circulating prolactin (PRL) levels are low during non-breeding times and significantly increase during late incubation and early post-hatch chick care. Because of this pattern, PRL has been suggested to be involved in the initiation of parental care in birds, but rarely has this hypothesis been causally tested. To begin testing the hypothesis, we inhibited the release of endogenous PRL with bromocriptine (BR) on the 3days prior to hatching in incubating parents and the first 2days of post-hatch care, when PRL was found to be highest in zebra finches. Nest temperatures were recorded during all 5days and parental behavior was recorded on days 1-2 post-hatch. In addition to hormonal systems, reproductive experience may also influence parental care; therefore, we tested age-matched inexperienced and experienced pairs in each group. BR either eliminated or drastically reduced chick brooding and feeding behavior, resulting in decreased nest temperatures on days 1 and 2 post-hatch. Experienced control birds fed chicks more than inexperienced birds and control females fed more than males. Chick feeding behavior was positively correlated in control male-female pairs, but not in BR pairs. This is one of the few causal studies to demonstrate that PRL is necessary for post-hatch care in a biparental songbird, and is the first to show this effect in zebra finches.

Early life manipulations of vasopressin-family peptides alter vocal learning.

Vocal learning from social partners is crucial for the successful development of communication in a wide range of species. Social interactions organize attention and enhance motivation to learn species-typical behaviour. However, the neurobiological mechanisms connecting social motivation and vocal learning are unknown. Using zebra finches (Taeniopygia guttata), a ubiquitous model for vocal learning, we show that manipulations of nonapeptide hormones in the vasopressin family (arginine vasotocin, AVT) early in development can promote or disrupt both song and social motivation. Young male zebra finches, like human infants, are socially gregarious and require interactive feedback from adult tutors to learn mature vocal forms. To investigate the role of social motivational mechanisms in song learning, in two studies, we injected hatchling males with AVT or Manning compound (MC, a nonapeptide receptor antagonist) on days 2-8 post-hatching and recorded song at maturity. In both studies, MC males produced a worse match to tutor song than controls. In study 2, which experimentally controlled for tutor and genetic factors, AVT males also learned song significantly better compared with controls. Furthermore, song similarity correlated with several measures of social motivation throughout development. These findings provide the first evidence that nonapeptides are critical to the development of vocal learning.

Developmental effects of vasotocin and nonapeptide receptors on early social attachment and affiliative behavior in the zebra finch.

Zebra finches demonstrate selective affiliation between juvenile offspring and parents, which, like affiliation between pair partners, is characterized by proximity, vocal communication and contact behaviors. This experiment tested the hypothesis that the nonapeptide arginine vasotocin (AVT, avian homologue of vasopressin) and nonapeptide receptors play a role prior to fledging in the development of affiliative behavior. Zebra finch hatchlings of both sexes received daily intracranial injections (post-hatch days 2-8) of either AVT, Manning Compound (MC, a potent V1a receptor antagonist) or saline (vehicle control). The social development of both sexes was assessed by measuring responsiveness to isolation from the family and subsequent reunion with the male parent after fledging. In addition, we assessed the changes in affiliation with the parents, unfamiliar males, and unfamiliar females each week throughout juvenile development. Compared to controls, MC subjects showed decreased attachment to the parents and MC males did not show the normal increase in affiliative interest in opposite sex individuals as they reached reproductive maturity. In contrast, AVT subjects showed a sustained affiliative interest in parents throughout development, and males showed increased interest in opposite sex conspecifics as they matured. These results provide the first evidence suggesting that AVT and nonapeptide receptors play organizational roles in social development in a bird.

Prolactin is related to individual differences in parental behavior and reproductive success in a biparental passerine, the zebra finch (Taeniopygia guttata).

Variation in parental care can lead to important fitness consequences. The endocrine system is known to regulate physiological and behavioral reproductive traits that are important contributors to lifetime reproductive success. However, the hormonal basis of variation in avian parental care is still not well understood. Plasma prolactin (PRL) concentrations are generally high during post-hatch parental care in birds, and may be a candidate mechanism that regulates variation in parental care and other reproductive success outcomes. Here we analyze the relationship between PRL, parental behavior (chick brooding and feeding) and reproductive success outcomes (clutch size, number of chicks hatched, and chick survival) for the first time in the zebra finch (Taeniopygia guttata). Birds were given cabergoline, a dopamine agonist traditionally used to lower prolactin in mammals, or vehicle in their food. Cabergoline had no effect on prolactin concentrations, but across both groups we found that PRL is positively correlated with parental behavior, number of chicks hatched, and chick survival, but not clutch size. Results from this study will inform hypotheses and predictions for future manipulation studies which test for a causal role for PRL in parental traits.

Relationship between prolactin, reproductive experience, and parental care in a biparental songbird, the zebra finch (Taeniopygia guttata).

Hormonal systems have long been thought to play an important role in stimulating the onset of parental behavior, a critical component of reproductive success in a variety of taxa. Elevations in the peptide hormone prolactin (PRL) have been repeatedly positively correlated with the onset and maintenance of parental care across vertebrate species. A causal role for PRL in parental care has been established in several mammalian species, but less evidence for a causal role of PRL and parental care exists in birds. The zebra finch, a socially monogamous, biparental songbird, is an exceptionally useful animal model to study parental care and other close social relationships. Both sexes share parental care equally, exhibit the same parental behaviors, and show a marked improvement in breeding success with experience. We hypothesize that PRL is critically involved in the expression of zebra finch parental care and predict that circulating PRL levels will increase with breeding experience. To begin testing this, we measured plasma PRL concentrations in 14 male-female zebra finch pairs (N=28) across two breeding cycles, using a repeated measures design. PRL was measured in the birds' first, reproductively inexperienced, breeding cycle beginning at courtship and extending through chick fledging. PRL was measured again during the birds' second, reproductively experienced, breeding cycle, beginning with egg laying until chick fledging. We found that plasma PRL is significantly elevated from non-breeding concentrations during late incubation and early post-hatch care and that this elevation is greater in the reproductively experienced cycle compared to the inexperienced cycle. Findings of this study will be used to inform hypotheses and predictions for future experimental manipulations of PRL during parental care.

Sexual dimorphism in body size and the origin of sex-determination systems.

Diverse sex-determining systems occur in vertebrates, including environmental sex determination (ESD), genetic sex determination (GSD) of type XX/XY (heterogametic males), and GSD of type ZZ/ZW (heterogametic females). The origins of the two genetic types are poorly understood. We use protected invasion theory to derive a model that generates testable predictions about the origins of the two genetic types from ESD. Protected invasion theory predicts biases in the evolutionary origins of new traits by focusing on the probability that a selectively favored trait will avoid loss by genetic drift when rare. We show that the theory makes predictions about the conditions under which XY or ZW systems are more likely to arise from an ancestral state of ESD. In particular, assuming that there is an average trend toward increasing body size in lineages, the origins of XY systems are predicted to be accompanied by increases in male∶female body size ratio. In contrast, ZW systems are predicted to be accompanied by decreases in male∶female body size ratio. We find support for these predictions in the form of a marked association among vertebrates between sex-determining system and body size dimorphism in paired comparisons independent of shared phylogeny.

Newly paired zebra finches have higher dopamine levels and immediate early gene Fos expression in dopaminergic neurons.

Most birds are socially monogamous, yet little is known about the neural pathways underlying avian monogamy. Recent studies have implicated dopamine as playing a role in courtship and affiliation in a socially monogamous songbird, the zebra finch (Taeniopygia guttata). In the present study, we sought to understand the specific contribution to pair formation in zebra finches of the mesolimbic dopaminergic pathway that projects from the midbrain ventral tegmental area to the nucleus accumbens. We observed that paired birds had higher levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid in the ventral medial striatum, where the nucleus accumbens is situated, than unpaired birds. Additionally, we found that the percentage of dopaminergic neurons expressing immediate early gene Fos, a marker of neuronal activity, was higher in the ventral tegmental area of paired birds than in that of unpaired birds. These data are consistent with a role for the mesolimbic dopaminergic pathway in pair formation in zebra finches, suggesting the possibility of a conserved neural mechanism of monogamy in birds and mammals.

Long-lasting and sex-specific consequences of elevated egg yolk testosterone for social behavior in Japanese quail.

In birds, early exposure to steroid hormones deposited in egg yolks is hypothesized to result in long-lasting effects on brain and behavior. However, the long-term effects of maternal androgens on the development of social behavior, and whether these could interfere with the effects of the endogenous gonadal hormones that mediate sexual differentiation, remain poorly known. To answer these questions, we enhanced yolk testosterone by injecting testosterone (T) in oil into Japanese quail (Coturnix japonica) eggs prior to incubation. Vehicle-injected (V) eggs served as controls. From age 3 weeks to 8 weeks, sexual development was measured using morphological and physiological traits, and social behavior was measured, including male-typical sexual behavior. In females, treatment with testosterone boosted growth. Males from T-injected eggs developed an affiliative preference for familiar females and differed from V-injected males in the acoustic features of their crows, whereas sexual interest (looking behavior) and copulatory behavior were not affected. These long-lasting and sex-specific yolk testosterone effects on the development of dimorphic traits, but without disrupting sexual differentiation of reproductive behavior suggest potential organizational effects of maternal testosterone, but acting through separate processes than the endocrine mechanisms previously shown to control sexual differentiation. Separate processes could reflect the action of androgens at different times or on multiple targets that are differentially sensitive to steroids or develop at different rates.

Maternal effects in quail and zebra finches: Behavior and hormones.

Maternal effects are influences of parents on offspring phenotype occurring through pathways other than inherited DNA. In birds, two important routes for such transmission are parental behavior and non-DNA egg constituents such as yolk hormones. Offspring traits subject to parental effects include behavior and endocrine function. Research from the Adkins-Regan lab has used three avian species to investigate maternal effects related to hormones and behavior. Experiments with chickens and Japanese quail have shown that maternal sex steroids can influence sex determination to produce biased offspring sex ratios. Because all birds have a ZZ/ZW chromosomal sex determining system in which the female parent determines the sex of the offspring, these results raise the possibility that maternal steroids can influence the outcome of sex chromosome meiosis. Learning has been shown to influence egg investment by female quail in ways that are likely to alter offspring phenotype. In quail, embryonic and exogenous sex steroids have well established and long-lasting effects on sexual differentiation of behavior during a critical period in ovo, but elevated yolk testosterone has long-term effects on behavior that do not seem to be occurring through an alteration in sexual differentiation. In biparental zebra finches, removal of mothers alters not only later behavior, but also the adult response of the hypothalamic-pituitary-adrenal (HPA) axis to an environmental stressor, as indicated by plasma corticosterone. Birds raised only by fathers have lower levels of mRNA for both glucocorticoid receptors in several brain regions as adults. These studies add to the evidence that one generation influences the behavioral or endocrine phenotype of the next through routes other than transmission of DNA. Additional research will be required to understand the adaptive significance of these effects.

Neuroendocrine contributions to sexual partner preference in birds.

A majority of birds are socially monogamous, providing exceptional opportunities to discover neuroendocrine mechanisms underlying preferences for opposite-sex partners where the sexes form extended affiliative relationships. Zebra finches have been the focus of the most systematic program of research to date in any socially monogamous animal. In this species, sexual partner preference can be partially or largely sex reversed with hormone manipulations during early development, suggesting a role for organizational hormone actions. This same conclusion emerges from research with Japanese quail, which do not form long-term pairs. In zebra finches, social experience manipulations during juvenile development also can sex reverse partner preference, either alone or in combination with an early hormone manipulation. Although there are several candidate brain regions where neural mechanisms could underlie these effects of hormones or social experience, the necessary research has not yet been done to determine their involvement. The neuroendocrinology of avian sexual partner preference is still frontier territory.

Deprivation of maternal care has long-lasting consequences for the hypothalamic-pituitary-adrenal axis of zebra finches.

Early-life stress caused by the deprivation of maternal care has been shown to have long-lasting effects on the hypothalamic-pituitary-adrenal (HPA) axis in offspring of uniparental mammalian species. We asked if deprivation of maternal care in biparental species alters stress responsiveness of offspring, using a biparental avian species--the zebra finch, Taeniopygia guttata. In our experiment, one group of birds was raised by both male and female parents (control), and another was raised by males alone (maternally deprived). During adulthood, offspring of both groups were subjected to two stressors (restraint and isolation), and corticosterone concentrations were measured. Additionally, we measured baseline levels of the two corticosteroid receptors--glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)--in the hippocampus, hypothalamus and cerebellum. Our results suggest that maternally deprived offspring are hyper-responsive to isolation in comparison with controls. Furthermore, mRNA levels of both GR and MR receptors are altered in maternally deprived offspring in comparison with controls. Thus, absence of maternal care has lasting consequences for HPA function in a biparental species where paternal care is available.

Hormonal organization and activation: evolutionary implications and questions.

Comparative endocrinology is a fascinating field of science in part because it addresses both ultimate and proximate causation. Research on sexual dimorphism and sexual differentiation has excellent potential for this kind of integration. Vertebrate comparative endocrinologists have made many important discoveries about the role of genes and sex steroid hormones in the organization and activation of sexually differentiated behavior, brain function, anatomy and physiology. In addition to taxonomically general principles and conserved features, there is also striking diversity in sexual differentiation processes. Much of the evolutionary basis of this diversity (its phylogenetic history and adaptive functions) is not well understood. A set of questions is raised to illustrate this point, with an emphasis on mechanisms of sexual dimorphism in body size and ornamentation, sexual differentiation of avian behavior, particularly in Japanese quail and zebra finches, and the puzzle of the phylogenetic distribution of vertebrate sex determining mechanisms. Applying a comparative approach grounded in established phylogenies and concepts from evolutionary developmental biology such as developmental modules holds promise for generating and testing new hypotheses and eventually answering some of these questions.

Effect of isolation and conspecific presence in a novel environment on corticosterone concentrations in a social avian species, the zebra finch (Taeniopygia guttata).

Zebra finches are a highly social and monogamous avian species. In the present study, we sought to determine the effect of social isolation (separation from the flock) in a novel environment with and without a conspecific present on the adrenocortical activity of paired and unpaired individuals of this species. With regard to paired birds, we hypothesized that the presence of the mate during isolation from the group would act as a social buffer against the stressful effects of isolation. We observed that 10 but not 30 minutes of social isolation resulted in elevated concentrations of corticosterone in unpaired and paired male zebra finches in comparison to baseline concentrations of corticosterone. Furthermore, the presence of a mate during isolation in a novel environment did not have a buffering effect against increases in corticosterone concentrations. Additionally, to compare concentrations of corticosterone in response to isolation (in a novel environment) to a previously well-established stressor, we subjected groups of birds to restraint. We observed that 10 or 30 minutes of restraint led to significantly higher concentrations of corticosterone as compared to baseline. Finally, to rule out the possibility that merely handling a bird would result in significantly elevated concentrations of corticosterone as compared to baseline samples, we measured corticosterone concentrations 10 or 30 minutes after handling involving capture and release only. Our results suggest that handling alone might have contributed to the elevation of corticosterone in birds exposed to 10 minutes but not 30 minutes of restraint. Handling by itself did not account, however, for the elevated corticosterone in birds socially isolated for 10 minutes.

Copulatory behaviors and body condition predict post-mating female hormone concentrations, fertilization success, and primary sex ratios in Japanese quail.

Environmental cues and social interactions are known to influence reproductive physiology and behavior in vertebrates. In female birds, male courtship displays can result in the growth of ovarian follicles, the production of reproductive hormones, and stimulation of oviduct development, all of which have the potential to influence maternal investment. Male Japanese quail follow a typical sequence of copulatory behaviors during a mating interaction and often force copulations with unreceptive females. We hypothesized that female Japanese quail could adjust maternal investment in response to male copulatory behaviors during a single mating interaction. We investigated the relationships between 1) male copulatory behaviors and post-mating concentrations of steroids in the female, 2) female steroid concentrations and fertilization success of inseminations and 3) female steroid concentrations and the offspring sex ratio. We found that male condition and copulatory behaviors predicted female steroid concentrations and maternal investment in eggs laid after a mating trial. The body condition of one or both mates was a significant predictor of the changes in female corticosterone and testosterone concentrations after mating, whereas specific male copulatory behaviors significantly predicted changes in female progesterone concentrations. Male and female body condition, male neck grabs and post-mating concentrations of female corticosterone, progesterone, and testosterone were all significant predictors of egg fertilization rates. Female body condition, male copulation efficiency, and female testosterone concentrations were significant predictors of offspring sex ratios. Our results show that phenotypic and behavioral characteristics of male Japanese quail modulate female steroid concentrations and result in changes in maternal investment.

Hormones and sexual differentiation of avian social behavior.

Research on the hormonal basis of sexual differentiation of mammalian behavior and its neural substrates constitutes an important chapter in the study of neural development that has led to important insights into how to conceptualize sexuality. Here research on sexual differentiation of avian social behavior is discussed with an eye toward potential insights into avian sexuality. Hormone manipulation experiments with Japanese quail (Coturnix japonica) and zebra finches (Taeniopygia guttata) reveal dissociations between different components of sexual and social behavior. None of the components of the male behavioral phenotype appear to be organized by androgens. Avian sexuality arises from multiple processes and pathways that appear to differ for mating behavior, sexual partner preference, and pairing. This provides the mechanistic bases for changes in social behavior in the course of avian evolution.