Transient sensorimotor projections in the developmental song learning period.

Memory recall and guidance are essential for motor skill acquisition. Like humans learning to speak, male zebra finches learn to sing by first memorizing and then matching their vocalization to the tutor's song (TS) during specific developmental periods. Yet, the neuroanatomical substrate supporting auditory-memory-guided sensorimotor learning has remained elusive. Here, using a whole-brain connectome analysis with activity-dependent viral expression, we identified a transient projection into the motor region, HVC, from neuronal ensembles responding to TS in the auditory forebrain, the caudomedial nidopallium (NCM), in juveniles. Virally induced cell death of the juvenile, but not adult, TS-responsive NCM neurons impaired song learning. Moreover, isolation, which delays closure of the sensory, but not the motor, learning period, did not affect the decrease of projections into the HVC from the NCM TS-responsive neurons after the song learning period. Taken together, our results suggest that dynamic axonal pruning may regulate timely auditory-memory-guided vocal learning during development.

Daily singing of adult songbirds functions to maintain song performance independently of auditory feedback and age.

Many songbirds learn to produce songs through vocal practice in early life and continue to sing daily throughout their lifetime. While it is well-known that adult songbirds sing as part of their mating rituals, the functions of singing behavior outside of reproductive contexts remain unclear. Here, we investigated this issue in adult male zebra finches by suppressing their daily singing for two weeks and examining the effects on song performance. We found that singing suppression decreased the pitch, amplitude, and duration of songs, and that those song features substantially recovered through subsequent free singing. These reversible song changes were not dependent on auditory feedback or the age of the birds, contrasting with the adult song plasticity that has been reported previously. These results demonstrate that adult song structure is not stable without daily singing, and suggest that adult songbirds maintain song performance by preventing song changes through physical act of daily singing throughout their life. Such daily singing likely functions as vocal training to maintain the song production system in optimal conditions for song performance in reproductive contexts, similar to how human singers and athletes practice daily to maintain their performance.

A predisposed motor bias shapes individuality in vocal learning.

The development of individuality during learned behavior is a common trait observed across animal species; however, the underlying biological mechanisms remain understood. Similar to human speech, songbirds develop individually unique songs with species-specific traits through vocal learning. In this study, we investigate the developmental and molecular mechanisms underlying individuality in vocal learning by utilizing F1 hybrid songbirds (Taeniopygia guttata cross with Taeniopygia bichenovii), taking an integrating approach combining experimentally controlled systematic song tutoring, unbiased discriminant analysis of song features, and single-cell transcriptomics. When tutoring with songs from both parental species, F1 hybrid individuals exhibit evident diversity in their acquired songs. Approximately 30% of F1 hybrids selectively learn either song of the two parental species, while others develop merged songs that combine traits from both species. Vocal acoustic biases during vocal babbling initially appear as individual differences in songs among F1 juveniles and are maintained through the sensitive period of song vocal learning. These vocal acoustic biases emerge independently of the initial auditory experience of hearing the biological father's and passive tutored songs. We identify individual differences in transcriptional signatures in a subset of cell types, including the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei, which are associated with variations of vocal acoustic features. These findings suggest that a genetically predisposed vocal motor bias serves as the initial origin of individual variation in vocal learning, influencing learning constraints and preferences.

Dosage compensation of Z sex chromosome genes in avian fibroblast cells.

In birds, sex is genetically determined; however, the molecular mechanism is not well-understood. The avian Z sex chromosome (chrZ) lacks whole chromosome inactivation, in contrast to the mammalian chrX. To investigate chrZ dosage compensation and its role in sex specification, we use a highly quantitative method and analyze transcriptional activities of male and female fibroblast cells from seven bird species. Our data indicate that three fourths of chrZ genes are strictly compensated across Aves, similar to mammalian chrX. We also present a complete list of non-compensated chrZ genes and identify Ribosomal Protein S6 (RPS6) as a conserved sex-dimorphic gene in birds.

Nicotinic acetylcholine receptors in a songbird brain.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission and cell signaling, which contribute to learning, memory, and the execution of motor skills. Birdsong is a complex learned motor skill in songbirds. Although the existence of 15 nAChR subunits has been predicted in the avian genome, their expression patterns and potential contributions to song learning and production have not been comprehensively investigated. Here, we cloned all the 15 nAChR subunits (ChrnA1-10, B2-4, D, and G) from the zebra finch brain and investigated the mRNA expression patterns in the neural pathways responsible for the learning and production of birdsong during a critical period of song learning. Although there were no detectable hybridization signals for ChrnA1, A6, A9, and A10, the other 11 nAChR subunits were uniquely expressed in one or more major subdivisions in the song nuclei of the songbird brain. Of these 11 subunits, ChrnA3-5, A7, and B2 were differentially regulated in the song nuclei compared with the surrounding anatomically related regions. ChrnA5 was upregulated during the critical period of song learning in the lateral magnocellular nucleus of the anterior nidopallium. Furthermore, single-cell RNA sequencing revealed ChrnA7 and B2 to be the major subunits expressed in neurons of the vocal motor nuclei HVC and robust nucleus of the arcopallium, indicating the potential existence of ChrnA7-homomeric and ChrnB2-heteromeric nAChRs in limited cell populations. These results suggest that relatively limited types of nAChR subunits provide functional contributions to song learning and production in songbirds.

Fetal blockade of nicotinic acetylcholine transmission causes autism-like impairment of biological motion preference in the neonatal chick.

Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects.

Seasonal regulation of singing-driven gene expression associated with song plasticity in the canary, an open-ended vocal learner.

Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc, Egr1, c-fos, Nr4a1, Sik1, Dusp6, and Gadd45ß) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.

Phylogeny and mechanisms of shared hierarchical patterns in birdsong.

Organizational patterns can be shared across biological systems, and revealing the factors shaping common patterns can provide insight into fundamental biological mechanisms. The behavioral pattern that elements with more constituents tend to consist of shorter constituents (Menzerath's law [ML]) was described first in speech and language (e.g., words with more syllables consist of shorter syllables) and subsequently in music and animal communication. Menzerath's law is hypothesized to reflect efficiency in information transfer, but biases and constraints in motor production can also lead to this pattern. We investigated the evolutionary breadth of ML and the contribution of production mechanisms to ML in the songs of 15 songbird species. Negative relationships between the number and duration of constituents (e.g., syllables in phrases) were observed in all 15 species. However, negative relationships were also observed in null models in which constituents were randomly allocated into observed element durations, and the observed negative relationship for numerous species did not differ from the null model; consequently, ML in these species could simply reflect production constraints and not communicative efficiency. By contrast, ML was significantly different from the null model for more than half the cases, suggesting additional organizational rules are imposed onto birdsongs. Production mechanisms are also underscored by the finding that canaries and zebra finches reared without auditory experiences that guide vocal development produced songs with nearly identical ML patterning as typically reared birds. These analyses highlight the breadth with which production mechanisms contribute to this prevalent organizational pattern in behavior.

Identification of novel avian and mammalian deltaviruses provides new insights into deltavirus evolution.

Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here, we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite-helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.

Manipulations of sensory experiences during development reveal mechanisms underlying vocal learning biases in zebra finches.

Biological predispositions in learning can bias and constrain the cultural evolution of social and communicative behaviors (e.g., speech and birdsong), and lead to the emergence of behavioral and cultural "universals." For example, surveys of laboratory and wild populations of zebra finches (Taeniopygia guttata) document consistent patterning of vocal elements ("syllables") with respect to their acoustic properties (e.g., duration, mean frequency). Furthermore, such universal patterns are also produced by birds that are experimentally tutored with songs containing randomly sequenced syllables ("tutored birds"). Despite extensive demonstrations of learning biases, much remains to be uncovered about the nature of biological predispositions that bias song learning and production in songbirds. Here, we examined the degree to which "innate" auditory templates and/or biases in vocal motor production contribute to vocal learning biases and production in zebra finches. Such contributions can be revealed by examining acoustic patterns in the songs of birds raised without sensory exposure to song ("untutored birds") or of birds that are unable to hear from early in development ("early-deafened birds"). We observed that untutored zebra finches and early-deafened zebra finches produce songs with positional variation in some acoustic features (e.g., mean frequency) that resemble universal patterns observed in tutored birds. Similar to tutored birds, early-deafened birds also produced song motifs with alternation in acoustic features across adjacent syllables. That universal acoustic patterns are observed in the songs of both untutored and early-deafened birds highlights the contribution motor production biases to the emergence of universals in culturally transmitted behaviors.

Statistical learning for vocal sequence acquisition in a songbird.

Birdsong is a learned communicative behavior that consists of discrete acoustic elements ("syllables") that are sequenced in a controlled manner. While the learning of the acoustic structure of syllables has been extensively studied, relatively little is known about sequence learning in songbirds. Statistical learning could contribute to the acquisition of vocal sequences, and we investigated the nature and extent of sequence learning at various levels of song organization in the Bengalese finch, Lonchura striata var. domestica. We found that, under semi-natural conditions, pupils (sons) significantly reproduced the sequence statistics of their tutor's (father's) songs at multiple levels of organization (e.g., syllable repertoire, prevalence, and transitions). For example, the probability of syllable transitions at "branch points" (relatively complex sequences that are followed by multiple types of transitions) were significantly correlated between the songs of tutors and pupils. We confirmed the contribution of learning to sequence similarities between fathers and sons by experimentally tutoring juvenile Bengalese finches with the songs of unrelated tutors. We also discovered that the extent and fidelity of sequence similarities between tutors and pupils were significantly predicted by the prevalence of sequences in the tutor's song and that distinct types of sequence modifications (e.g., syllable additions or deletions) followed distinct patterns. Taken together, these data provide compelling support for the role of statistical learning in vocal production learning and identify factors that could modulate the extent of vocal sequence learning.

Transcriptional regulatory divergence underpinning species-specific learned vocalization in songbirds.

Learning of most motor skills is constrained in a species-specific manner. However, the proximate mechanisms underlying species-specific learned behaviors remain poorly understood. Songbirds acquire species-specific songs through learning, which is hypothesized to depend on species-specific patterns of gene expression in functionally specialized brain regions for vocal learning and production, called song nuclei. Here, we leveraged two closely related songbird species, zebra finch, owl finch, and their interspecific first-generation (F1) hybrids, to relate transcriptional regulatory divergence between species with the production of species-specific songs. We quantified genome-wide gene expression in both species and compared this with allele-specific expression in F1 hybrids to identify genes whose expression in song nuclei is regulated by species divergence in either cis- or trans-regulation. We found that divergence in transcriptional regulation altered the expression of approximately 10% of total transcribed genes and was linked to differential gene expression between the two species. Furthermore, trans-regulatory changes were more prevalent than cis-regulatory and were associated with synaptic formation and transmission in song nucleus RA, the avian analog of the mammalian laryngeal motor cortex. We identified brain-derived neurotrophic factor (BDNF) as an upstream mediator of trans-regulated genes in RA, with a significant correlation between individual variation in BDNF expression level and species-specific song phenotypes in F1 hybrids. This was supported by the fact that the pharmacological overactivation of BDNF receptors altered the expression of its trans-regulated genes in the RA, thus disrupting the learned song structures of adult zebra finch songs at the acoustic and sequence levels. These results demonstrate functional neurogenetic associations between divergence in region-specific transcriptional regulation and species-specific learned behaviors.

Corticobasal ganglia projecting neurons are required for juvenile vocal learning but not for adult vocal plasticity in songbirds.

Birdsong, like human speech, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. However, it is unknown how the connections between cortical and basal ganglia components contribute to vocal motor skill learning, as mammalian motor cortices serve multiple types of motor action and most experimentally tractable animals do not exhibit vocal learning. Here, we leveraged the zebra finch, a songbird, as an animal model to explore the function of the connectivity between cortex-like (HVC) and basal ganglia (area X), connected by HVC(X) projection neurons with temporally precise firing during singing. By specifically ablating HVC(X) neurons, juvenile zebra finches failed to copy tutored syllable acoustics and developed temporally unstable songs with less sequence consistency. In contrast, HVC(X)-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results indicate that the corticobasal ganglia input is important for learning the acoustic and temporal aspects of song structure, but not for generating vocal fluctuations that contribute to the maintenance of an already learned vocal pattern.

Auditory-Motor Matching in Vocal Recognition and Imitative Learning.

Songbirds possess mirror neurons (MNs) activating during the perception and execution of specific features of songs. These neurons are located in high vocal center (HVC), a premotor nucleus implicated in song perception, production and learning, making worth to inquire their properties and functions in vocal recognition and imitative learning. By integrating a body of brain and behavioral data, we discuss neurophysiology, anatomical, computational properties and possible functions of songbird MNs. We state that the neurophysiological properties of songbird MNs depends on sensorimotor regions that are outside the auditory neural system. Interestingly, songbirds MNs can be the result of the specific type of song representation possessed by some songbird species. At the functional level, we discuss whether songbird MNs are involved in others' song recognition, by dissecting the function of recognition in various different but possible overlapping processes: action-oriented perception, discriminative-oriented perception and identification of the signaler. We conclude that songbird MNs may be involved in recognizing other singer's vocalizations, while their role in imitative learning still require to solve how auditory feedback are used to correct own vocal performance to match the tutor song. Finally, we compare songbird and human mirror responses, hypothesizing a case of convergent evolution, and proposing new experimental directions.

Inter- and intra-specific differences in muscarinic acetylcholine receptor expression in the neural pathways for vocal learning in songbirds.

Acetylcholine receptors (AChRs) abound in the central nervous system of vertebrates. Muscarinic AChRs (mAChRs), a functional subclass of AChRs, mediate neuronal responses via intracellular signal transduction. They also play roles in sensorimotor coordination and motor skill learning by enhancing cortical plasticity. Learned birdsong is a complex motor skill acquired through sensorimotor coordination during a critical period. However, the functions of AChRs in the neural circuits for vocal learning and production remain largely unexplored. Here, we report the unique expression of mAChRs subunits (chrm2-5) in the song nuclei of zebra finches. The expression of excitatory subunits (chrm3 and chrm5) was downregulated in the song nuclei compared with the surrounding brain regions. In contrast, the expression of inhibitory mAChRs (chrm2 and chrm4) was upregulated in the premotor song nucleus HVC relative to the surrounding nidopallium. Chrm4 showed developmentally different expression in HVC during the critical period. Compared with chrm4, individual differences in chrm2 expression emerged in HVC early in the critical period. These individual differences in chrm2 expression persisted despite testosterone administration or auditory deprivation, which altered the timing of song stabilization. Instead, the variability in chrm2 expression in HVC correlated with parental genetics. In addition, chrm2 expression in HVC exhibited species differences and individual variability among songbird species. These results suggest that mAChRs play an underappreciated role in the development of species and individual differences in song patterns by modulating the excitability of HVC neurons, providing a potential insight into the gating of auditory responses in HVC neurons.

Vocal practice regulates singing activity-dependent genes underlying age-independent vocal learning in songbirds.

The development of highly complex vocal skill, like human language and bird songs, is underlain by learning. Vocal learning, even when occurring in adulthood, is thought to largely depend on a sensitive/critical period during postnatal development, and learned vocal patterns emerge gradually as the long-term consequence of vocal practice during this critical period. In this scenario, it is presumed that the effect of vocal practice is thus mainly limited by the intrinsic timing of age-dependent maturation factors that close the critical period and reduce neural plasticity. However, an alternative, as-yet untested hypothesis is that vocal practice itself, independently of age, regulates vocal learning plasticity. Here, we explicitly discriminate between the influences of age and vocal practice using a songbird model system. We prevented zebra finches from singing during the critical period of sensorimotor learning by reversible postural manipulation. This enabled to us to separate lifelong vocal experience from the effects of age. The singing-prevented birds produced juvenile-like immature song and retained sufficient ability to acquire a tutored song even at adulthood when allowed to sing freely. Genome-wide gene expression network analysis revealed that this adult vocal plasticity was accompanied by an intense induction of singing activity-dependent genes, similar to that observed in juvenile birds, rather than of age-dependent genes. The transcriptional changes of activity-dependent genes occurred in the vocal motor robust nucleus of the arcopallium (RA) projection neurons that play a critical role in the production of song phonology. These gene expression changes were accompanied by neuroanatomical changes: dendritic spine pruning in RA projection neurons. These results show that self-motivated practice itself changes the expression dynamics of activity-dependent genes associated with vocal learning plasticity and that this process is not tightly linked to age-dependent maturational factors.

Recurrent development of song idiosyncrasy without auditory inputs in the canary, an open-ended vocal learner.

Complex learned behaviors, like bird song and human speech, develop under the influence of both genetic and environmental factors. Accordingly, learned behaviors comprise species specificity and individual variability. Auditory information plays a critical role in vocal learning by songbirds, both to memorize tutor songs and to monitor own vocalizations. Nevertheless, audition-deprived songbirds develop structured, species-specific song patterns. It remains to be elucidated how the auditory input contributes to the development of individual variability of song characteristics. Here we show that an open-ended vocal learner, the canary, annually recapitulates individually unique songs without audition. Although the total number of syllable types was reduced by auditory deprivation, other vocal phenotypes examined in the syllable, phrase, and syntax of songs were conserved between the 1st and 2nd years, both in deafened and intact birds. In deafened canaries, approximately 60% of the syllables were yearly reproduced with consistent acoustic features, whereas the remaining syllables were replaced with new ones in an annual cycle of song development. These results indicate that the open-ended vocal learning of canaries involves an audition-independent mechanism for the development of recurrent song idiosyncrasy.

Singing activity-driven Arc expression associated with vocal acoustic plasticity in juvenile songbird.

Learned vocalization, including birdsong and human speech, is acquired through self-motivated vocal practice during the sensitive period of vocal learning. The zebra finch (Taeniopygia guttata) develops a song characterized by vocal variability and crystallizes a defined song pattern as adulthood. However, it remains unknown how vocal variability is regulated with diurnal singing during the sensorimotor learning period. Here, we investigated the expression of activity-dependent neuroplasticity-related gene Arc during the early plastic song phase to examine its potential association with vocal plasticity. We first confirmed that multiple acoustic features of syllables in the plastic song were dramatically and simultaneously modulated during the first 3 hr of singing in a day and the altered features were maintained until sleep. In a concurrent manner, Arc was intensely induced during morning singing and a subsequent attenuation during afternoon singing in the robust nucleus of the arcopallium (RA) and the interfacial nucleus of the nidopallium (NIf). The singing-driven Arc expression was not altered by circadian rhythm, but rather reduced during the day as juveniles produced more songs. Song stabilization accelerated by testosterone administration in juveniles was accompanied with attenuation of Arc induction in RA and NIf. In contrast, although early-deafened birds produced highly unstable song even at adulthood, singing-driven Arc expression was not different between intact and early-deafened adults. These results suggest a potential functional link between Arc expression in RA and NIf and vocal plasticity during the sensorimotor phase of song learning. Nonetheless, Arc expression did not reflect the quality of bird's own song or auditory feedback.

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.

Sex Differences in Brain Thyroid Hormone Levels during Early Post-Hatching Development in Zebra Finch (Taeniopygia guttata).

Thyroid hormones are closely linked to the hatching process in precocial birds. Previously, we showed that thyroid hormones in brain had a strong impact on filial imprinting, an early learning behavior in newly hatched chicks; brain 3,5,3'-triiodothyronine (T3) peaks around hatching and imprinting training induces additional T3 release, thus, extending the sensitive period for imprinting and enabling subsequent other learning. On the other hand, blood thyroid hormone levels have been reported to increase gradually after hatching in altricial species, but it remains unknown how the brain thyroid hormone levels change during post-hatching development of altricial birds. Here, we determined the changes in serum and brain thyroid hormone levels of a passerine songbird species, the zebra finch using radioimmunoassay. In the serum, we found a gradual increase in thyroid hormone levels during post-hatching development, as well as differences between male and female finches. In the brain, there was clear surge in the hormone levels during development in males and females coinciding with the time of fledging, but the onset of the surge of thyroxine (T4) in males preceded that of females, whereas the onset of the surge of T3 in males succeeded that of females. These findings provide a basis for understanding the functions of thyroid hormones during early development and learning in altricial birds.