Zebra Finches Research Articles

Concave-to-convex curve conversion of fiber cells correlates with Y-shaped suture formation at the poles of the rodent lens

The eye lens contains convexly curved fiber cells that align in concentric layers around the lens anterior-posterior pole axis. For lens fiber differentiation at the equator, cells elongate with their apical and basal tips migrating towards the anterior and posterior poles, respectively. At each pole, the fiber tips meet opposing tips of other fiber cells, to form a suture. Although umbilical or point sutures are observed in fish and birds, line, Y- or star-shaped sutures are detected in other vertebrate lenses. Sutures that do not converge at the point are thought to result from intricate movements of the fiber tips, rather than a straightforward migration along a meridional path. The triggers that give rise to these variations are currently not understood. Our findings revealed that in the mouse embryo, the early-stage lens contains only concave curved fibers, and later, a zone of concave-to-convex curve conversion develops. At this point, a nascent suture in a linear shape appears at the posterior pole and subsequently progresses into a V-shape. This V-shape appears to further develop into a Y-shape as a branch extends from the apex of the V-shape. In lens of zebrafish and Xenopus larvae that form point sutures, this curve-conversion zone is not observed. In lens of adult birds (e.g. zebra finch) that form a point suture, these too also lack a curve-conversion zone. In our previous studies, we demonstrated that murine lens fibers undergoing curve conversion extend membrane protrusions, or lamellipodia, at their basal membranes. In line with this, we did not observe protrusions at the basal tips of fibers in the non-mammalian lenses of zebrafish, Xenopus, and zebra finch in which curve conversion does not occur. We propose that the concave-to-convex conversion in rodent lenses introduces defined paths for fiber cell tips, leading to a more elaborate and complex suture formation, compared to the simple point suture of lower vertebrates.

Temperature and water availability induce chronic stress responses in Zebra finch (Taeniopygia guttata).

Animals initiate physiological mechanisms to re-establish homeostasis following environmental stress. To understand how bird physiology responds to abiotic stress, we quantified changes in hematological markers of chronic stress response and body condition of male zebra finches (Taeniopygia guttata) acclimated for 18 weeks to hot and cool temperatures (daytime temperature: 40°C and 23°C) with water available ad libitum or restricted during half of the active phase. Ambient temperature induced greater chronic stress than restricted water availability. While cool compared to hot temperatures induced higher numbers of heterophils and H : L ratios and declined total leucocyte counts, water restriction decreased the number of lymphocytes compared to water ad libitum. Body condition correlated with hematological parameters showing that birds with better condition had greater capacity to face environmental stress. Therefore, prolonged exposure to cool periods may result in chronic stress in zebra finches, especially, if body condition is weakened.

Local sleep in songbirds: different simultaneous sleep states across the avian pallium.

Wakefulness and sleep have often been treated as distinct and global brain states. However, an emerging body of evidence on the local regulation of sleep stages challenges this conventional view. Apart from unihemispheric sleep, the current data that support local variations of neural oscillations during sleep are focused on the homeostatic regulation of local sleep, i.e., the role preceding awake activity. Here, to examine local differences in brain activity during natural sleep, we recorded the electroencephalogram and the local field potential across multiple sites within the avian pallium of zebra finches without perturbing the previous awake state. We scored the sleep stages independently in each pallial site and found that the sleep stages are not pallium-wide phenomena but rather deviate widely across electrode sites. Importantly, deeper electrode sites had a dominant role in defining the temporal aspects of sleep state congruence. Altogether, these findings show that local regulation of sleep oscillations also occurs in the avian brain without prior awake recruitment of specific pallial circuits and in the absence of mammalian cortical neural architecture.

Female calls promote song learning in male juvenile zebra finches

Social interactions promote vocal learning, but the impact of social feedback on this process and its neural circuitry is not well understood. We studied song imitation in juvenile male zebra finches raised either in the presence or absence of adult females. Juveniles learned songs more accurately with a female present, suggesting her presence improves imitation. When female calls correlated with practice, tutees’ songs better resembled the tutor’s, hinting toward the possibility that females provide practice-specific vocalizations. Intracellular recordings of HVC projection neurons revealed that a subset of these neurons in both juveniles and adults is sensitive to female calls during listening, suggesting a consistent neural mechanism for processing important vocalizations, regardless of age. However, call-related neural responses during singing were observed only in juveniles. These findings highlight how vocalizations, beyond those of the tutor, influence the neural circuits for vocal learning and production.

Auditory pallial regulation of the social behavior network

Sensory cues such as vocalizations contain important social information. Processing social features of vocalizations (e.g., vocalizer identity, emotional state) necessitates unpacking the complex sound streams in song or speech; this depends on circuits in pallial cortex. But whether and how this information is then transferred to limbic and hypothalamic regions remains a mystery. Here, using gregarious, vocal songbirds (female Zebra finches), we identify a prominent influence of the auditory pallium on one specific node of the Social Behavior Network, the lateral ventromedial nucleus of the hypothalamus (VMHl). Electrophysiological recordings revealed that social and non-social auditory stimuli elicited stimulus-specific spike trains that permitted stimulus differentiation in a large majority of VMHl single units, while transient disruption of auditory pallium elevated immediate early gene activity in VMHl. Descending functional connections such as these may be critical for the range of vertebrate species that rely on nuanced communication signals to guide social decision-making.

Breeding zebra finches prioritize reproductive bout over self-maintenance under food restriction.

Reproduction requires high amounts of energy, and challenging environments during breeding can force parents to prioritize their current reproductive bout over self-maintenance or vice versa. However, little is known about how common stressors, such as food restriction, can influence these trade-offs during breeding, and the physiological mechanisms for these trade-off decisions. In this study, adult zebra finches (Taeniopygia castanotis) were subjected to a control diet (ad libitum) or a 40% food restriction while raising nestlings and fledglings, and we measured body mass, furculum fat, plasma corticosterone (CORT) and blood glucose levels of the parents at the time of pairing, when their offspring fledged, and when their offspring reached nutritional independence. Offspring were also measured in body mass and growth rate from hatching until the end of the treatment period. Food-restricted parents had lower body mass when their offspring fledged and reached nutritional independence and higher baseline CORT at fledging compared to controls. Offspring did not differ in body mass or growth rate between treatment groups. However, there was no effect of food restriction on parents' furculum fat, baseline glucose, the adrenocortical response, or the glucose response. Furthermore, path analysis results suggest that alterations in baseline glucose is the primary driver of changes in body mass in parents and offspring brood mass. Taken together, these results suggest that food restriction during chick rearing in a short-lived passerine drives parents to prioritize their current reproductive bout over self-maintenance, and glucose could potentially be a mechanism for diverting energy toward parental effort.

Characterization and distribution of de novo mutations in the zebra finch

Germline de novo mutations (DNMs) provide the raw material for evolution. The DNM rate varies considerably between species, sexes and chromosomes. Here, we identify DNMs in the zebra finch (Taeniopygia guttata) across 16 parent-offspring trios using two genome assemblies of different quality. Using an independent genotyping assay, we validate 82% of the 150 candidate DNMs. DNM rates are consistent between both assemblies, with estimates of 6.14 × 10–9 and 6.36 × 10–9 per site per generation. We observe a strong paternal bias in DNM rates (male-to-female ratio ɑ ≈ 4), but this bias is in transition mutations only, leading to a transition-to-transversion ratio of 3.18 and 3.57. Finally, we find that DNMs tend to be randomly distributed across chromosomes, not associated with recombination hotspots or genic regions. However, the sex chromosome chrZ shows a roughly fourfold increased DNM rate compared to autosomes, which is more than the expected increase due to chrZ spending two-thirds of its time in males. Overall, our results further enhance our understanding of DNMs in passerine songbirds.

Single-cell RNA sequencing reveals surface markers of primordial germ cells in chicken and zebra finch.

Primordial germ cells (PGCs) in avian species exhibit unique developmental features, including the ability to migrate through the bloodstream and colonize the gonads, allowing their isolation at various developmental stages. Several methods have been developed for the isolation of avian PGCs, including density gradient centrifugation, size-dependent separation, and magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) using a stage-specific embryonic antigen-1 (SSEA-1) antibody. However, these methods present limitations in terms of efficiency and applicability across development stages. In particular, the specificity of SSEA-1 decreases in later developmental stages. Furthermore, surface markers that can be utilized for isolating or utilizing PGCs are lacking for wild birds, including zebra finches, and endangered avian species. To address this, we used single-cell RNA sequencing (scRNA-seq) to uncover novel PGC-specific surface markers in chicken and zebra finch. We screened for genes that were primarily expressed in the PGC population within the gonadal cells. Analyses of gene expression patterns and levels based on scRNA-seq, coupled with validation by RT-PCR, identified NEGR1 and SLC34A2 as novel PGC-specific surface markers in chickens and ESYT3 in zebra finches. Notably, these newly identified genes exhibited sustained expression not only during later developmental stages but also in reproductive tissues.

Early Oxidative Stress May Prevent a Red Ornament From Signaling Longevity.

Harsh early environmental conditions can exert delayed, long-lasting effects on phenotypes, including reproductive traits such as sexual signals. Indeed, adverse early conditions can accelerate development, increasing oxidative stress that may, in turn, impact adult sexual signals. Among signals, colorations produced by red ketocarotenoids seem to depend on mitochondrial functioning. Hence, they could reveal individual cell respiration efficiency. It has been hypothesized that these traits are unfalsifiable "index" signals of condition due to their deep connection to individual metabolism. Since mitochondrial dysfunction is frequently linked to aging, red ketocarotenoid-based ornaments could also be good signals of a critical fitness component: longevity. We tested this red color per longevity correlation in captive zebra finches. In addition, we experimentally decreased the synthesis of glutathione (a critical intracellular antioxidant) during the first days of the birds' life to resemble harsh early environmental conditions (e.g., undernutrition). Longevity was recorded until the death of the last bird (almost 9 years). Males, but not females, exhibiting a redder bill in early adulthood lived longer than males with paler bills, which agrees with some precedent studies. However, such bill redness-longevity connection was absent among males with inhibited glutathione synthesis. These findings may suggest that environmental factors can alter the reliability of red ketocarotenoid-based sexual signals, making them less unfalsifiable than believed.

Getting slimmer without dieting or exercising: zebra finches can rapidly lose mass while maintaining food intake and decreasing activity.

It is widely accepted that birds can adaptively regulate body mass in different ecological contexts, but little is known about how birds monitor and interpret their body mass or the mechanisms that allow for rapid changes in mass. Using captive zebra finches (Taeniopygia guttata), we experimentally increased perceived mass via attachment of weighted backpacks and provided birds with either an ad libitum mixed-seed diet or supplementary high-fat diet to investigate: (1) how birds assess their own body mass and (2) the physiological and/or behavioral mechanisms birds may employ to rapidly adjust body mass. In both experiments, and independent of diet treatment, birds with weighted backpacks rapidly lost mass within 2 days of backpack attachment while reducing overall activity and maintaining food intake. Additionally, our data suggest that birds interpret body mass via a physical mechanosensory pathway rather than a physiological pathway: rapid loss of mass between days 0 and 2 was not linked to changes in plasma metabolites (glycerol or triglyceride concentrations). We found no evidence that mass loss was a consequence of stress associated with attachment of weighted backpacks (based on plasma corticosterone measures). Our results suggest that the processes of energy balance and mass regulation involve a greater array of mechanisms than simply matching 'energy in', through the amount of food consumed, to 'energy out', dictated by activity. Zebra finches were able to decrease body mass through other, unidentified, mechanisms even while maintaining dietary intake and reducing overall activity.

Social and auditory experience shapes forebrain responsiveness in zebra finches before the sensitive period of vocal learning.

Early-life experiences with signals used in communication are instrumental in shaping an animal's social interactions. In songbirds, which use vocalizations for guiding social interactions and mate choice, recent studies show that sensory effects on development occur earlier than previously expected, even in embryos and nestlings. Here, we explore the neural dynamics underlying experience-dependent song categorization in young birds prior to the traditionally studied sensitive period of vocal learning that begins around 3 weeks post-hatch. We raised zebra finches either with their biological parents, cross-fostered by Bengalese finches beginning at embryonic day 9, or by only the non-singing mother from 2 days post-hatch. Then, 1-5 days after fledging, we conducted behavioral experiments and extracellular recordings in the auditory forebrain to test responses to zebra finch and Bengalese finch songs. Auditory forebrain neurons in cross-fostered and isolate birds showed increases in firing rate and decreases in responsiveness and selectivity. In cross-fostered birds, decreases in responsiveness and selectivity relative to white noise were specific to conspecific song stimuli, which paralleled behavioral attentiveness to conspecific songs in those same birds. This study shows that auditory and social experience can already impact song 'type' processing in the brains of nestlings, and that brain changes at this age can portend the effects of natal experience in adults.

Conservation of mutation and recombination parameters between mammals and zebra finch.

Most of our understanding of the fundamental processes of mutation and recombination stems from a handful of disparate model organisms and pedigree studies of mammals, with little known about other vertebrates. To gain a broader comparative perspective, we focused on the zebra finch (Taeniopygia castanotis), which, like other birds, differs from mammals in its karyotype (which includes many micro-chromosomes), in the mechanism by which recombination is directed to the genome, and in aspects of ontogenesis. We collected genome sequences from three generation pedigrees that provide information about 80 meioses, inferring 202 single-point de novo mutations, 1,174 crossovers, and 275 non-crossovers. On that basis, we estimated a sex-averaged mutation rate of 5.0 × 10-9 per base pair per generation, on par with mammals that have a similar generation time. Also as in mammals, we found a paternal germline mutation bias at later stages of gametogenesis (of 1.7 to 1) but no discernible difference between sexes in early development. We also examined recombination patterns, and found that the sex-averaged crossover rate on macro-chromosomes (1.05 cM/Mb) is again similar to values observed in mammals, as is the spatial distribution of crossovers, with a pronounced enrichment near telomeres. In contrast, non-crossover rates are more uniformly distributed. On micro-chromosomes, sex-averaged crossover rates are substantially higher (4.21 cM/Mb), as expected from crossover homeostasis, and both crossover and non-crossover events are more uniformly distributed. At a finer scale, recombination events overlap CpG islands more often than expected by chance, as expected in the absence of PRDM9. Despite differences in the mechanism by which recombination events are specified and the presence of many micro-chromosomes, estimates of the degree of GC-biased gene conversion (59%), the mean non-crossover conversion tract length (~23 bp), and the non-crossover to crossover ratio (6.7:1) are all comparable to those reported in primates and mice. The conservation of mutation and recombination properties from zebra finch to mammals suggest that these processes have evolved under stabilizing selection.

A behavioral analysis system MCFBM enables objective inference of songbirds’ attention during social interactions

Understanding animal behavior is crucial in behavioral neuroscience, aiming to unravel the mechanisms driving these behaviors. A significant milestone in this field is the analysis of behavioral reactions during social interactions. Despite their importance in social learning, the behavioral aspects of these interaction are not well understood in detail due to the lack of appropriate tools. We introduce a high-precision, marker-based motion-capture system for analyzing behavior in songbirds, accurately tracking body location and head direction in multiple freely moving finches during social interaction. Focusing on zebra finches, our analysis revealed variations in eye use based on individuals presented. We also observed behavioral changes during virtual and live presentations and a conditioned-learning paradigm. Additionally, the system effectively analyzed social interactions among mice. This system provides an efficient tool for advanced behavioral analysis in small animals and offers an objective method to infer their focus of attention.

Female zebra finches prefer partner's song despite absence of male voices when young

Female zebra finches prefer partner's song despite absence of male voices when young

Disinhibition enables vocal repertoire expansion after a critical period

The efficiency of motor skill acquisition is age-dependent, making it increasingly challenging to learn complex manoeuvres later in life. Zebra finches, for instance, acquire a complex vocal motor programme during a developmental critical period after which the learned song is essentially impervious to modification. Although inhibitory interneurons are implicated in critical period closure, it is unclear whether manipulating them can reopen heightened motor plasticity windows. Using pharmacology and a cell-type specific optogenetic approach, we manipulated inhibitory neuron activity in a premotor area of adult zebra finches beyond their critical period. When exposed to auditory stimulation in the form of novel songs, manipulated birds added new vocal syllables to their stable song sequence. By lifting inhibition in a premotor area during sensory experience, we reintroduced vocal plasticity, promoting an expansion of the syllable repertoire without compromising pre-existing song production. Our findings provide insights into motor skill learning capacities, offer potential for motor recovery after injury, and suggest avenues for treating neurodevelopmental disorders involving inhibitory dysfunctions.

Sex-specific effects of low-dose of acetamiprid on corticosterone levels but not on oxidative stress in House sparrows

Neonicotinoid insecticides are widely used in agriculture and have been linked to various detrimental physiological effects on wild birds. Despite this, the impact of acetamiprid – a less studied member of the neonicotinoid family – on the hypothalamic-pituitary-adrenal axis responsible for the hormonal regulation of the response to stress has rarely been examined in birds. In our study, we explored the effects of acetamiprid on feather levels of corticosterone, the major end product of the HPA, and blood oxidative status of House sparrows (Passer domesticus), following the ingestion of a low, field-realistic dose during two consecutive experiments in 2015 and 2016. We involved 112 birds in each experiment – 56 males and 56 females – that were administered a placebo or a dose of acetamiprid equivalent to 0.5% of the LD50 of the Zebra finch over the entire duration of the experiments, which lasted approximately three weeks. We measured corticosterone concentrations in feathers grown during an acclimation phase before ingestion and in newly grown feather after the experiment and assessed three oxidative stress markers in the blood. We found no impact of acetamiprid on oxidative stress markers. However, in 2015, male sparrows that ingested acetamiprid exhibited higher corticosterone levels in their feathers compared to those that received a placebo. No such difference was found in females. Interestingly, this effect was not observed in year 2016, which was characterised by less stressful conditions for the birds. These findings offer the first evidence of a potential effect of acetamiprid on corticosterone levels in a songbird, suggesting that ingesting this compound at very low dose may alter the endocrine physiology of the response to stress.

Pronounced early differentiation underlies zebra finch gonadal germ cell development

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken (Gallus gallus). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch (Taeniopygia castanotis), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

AVN: A Deep Learning Approach for the Analysis of Birdsong.

Deep learning tools for behavior analysis have enabled important new insights and discoveries in neuroscience. Yet, they often compromise interpretability and generalizability for performance, making it difficult to quantitively compare phenotypes across datasets and research groups. We developed a novel deep learning-based behavior analysis pipeline, Avian Vocalization Network (AVN), for the learned vocalizations of the most extensively studied vocal learning model species - the zebra finch. AVN annotates songs with high accuracy across multiple animal colonies without the need for any additional training data and generates a comprehensive set of interpretable features to describe the syntax, timing, and acoustic properties of song. We use this feature set to compare song phenotypes across multiple research groups and experiments, and to predict a bird's stage in song development. Additionally, we have developed a novel method to measure song imitation that requires no additional training data for new comparisons or recording environments, and outperforms existing similarity scoring methods in its sensitivity and agreement with expert human judgements of song similarity. These tools are available through the open-source AVN python package and graphical application, which makes them accessible to researchers without any prior coding experience. Altogether, this behavior analysis toolkit stands to facilitate and accelerate the study of vocal behavior by enabling a standardized mapping of phenotypes and learning outcomes, thus helping scientists better link behavior to the underlying neural processes.

Resilience of A Learned Motor Behavior After Chronic Disruption of Inhibitory Circuits.

Maintaining motor behaviors throughout life is crucial for an individual's survival and reproductive success. The neuronal mechanisms that preserve behavior are poorly understood. To address this question, we focused on the zebra finch, a bird that produces a highly stereotypical song after learning it as a juvenile. Using cell-specific viral vectors, we chronically silenced inhibitory neurons in the pre-motor song nucleus called the high vocal center (HVC), which caused drastic song degradation. However, after producing severely degraded vocalizations for around 2 months, the song rapidly improved, and animals could sing songs that highly resembled the original. In adult birds, single-cell RNA sequencing of HVC revealed that silencing interneurons elevated markers for microglia and increased expression of the Major Histocompatibility Complex I (MHC I), mirroring changes observed in juveniles during song learning. Interestingly, adults could restore their songs despite lesioning the lateral magnocellular nucleus of the anterior neostriatum (LMAN), a brain nucleus crucial for juvenile song learning. This suggests that while molecular mechanisms may overlap, adults utilize different neuronal mechanisms for song recovery. Chronic and acute electrophysiological recordings within HVC and its downstream target, the robust nucleus of the archistriatum (RA), revealed that neuronal activity in the circuit permanently altered with higher spontaneous firing in RA and lower in HVC compared to control even after the song had fully recovered. Together, our findings show that a complex learned behavior can recover despite extended periods of perturbed behavior and permanently altered neuronal dynamics. These results show that loss of inhibitory tone can be compensated for by recovery mechanisms partly local to the perturbed nucleus and do not require circuits necessary for learning.

Note similarities affect syntactic stability in zebra finches.

The acquisition of an acoustic template is a fundamental component of vocal imitation learning, which is used to refine innate vocalizations and develop a species-specific song. In the absence of a model, birds fail to develop species typical songs. In zebra finches (Taeniopygia guttata), tutored birds produce songs with a stereotyped sequence of distinct acoustic elements, or notes, which form the song motif. Songs of untutored individuals feature atypical acoustic and temporal structure. Here we studied songs and associated respiratory patterns of tutored and untutored male zebra finches to investigate whether similar acoustic notes influence the sequence of song elements. A subgroup of animals developed songs with multiple acoustically similar notes that are produced with alike respiratory motor gestures. These birds also showed increased syntactic variability in their adult motif. Sequence variability tended to occur near song elements which showed high similarity in acoustic structure and underlying respiratory motor gestures. The duration and depth of the inspirations preceding the syllables where syntactic variation occurred did not allow prediction of the following sequence of notes, suggesting that the varying duration and air requirement of the following expiratory pulse is not predictively encoded in the motor program. This study provides a novel method for calculation of motor/acoustic similarity, and the results of this study suggest that the note is a fundamental acoustic unit in the organization of the motif and could play a role in the neural code for song syntax.