Auditory discrimination learning and acoustic cue weighing in female zebra finches with localized FoxP1 knockdowns.

Rare disruptions of the transcription factor FOXP1 are implicated in a human neurodevelopmental disorder characterized by autism and/or intellectual disability with prominent problems in speech and language abilities. Avian orthologues of this transcription factor are evolutionarily conserved and highly expressed in specific regions of songbird brains, including areas associated with vocal production learning and auditory perception. Here, we investigated possible contributions of FoxP1 to song discrimination and auditory perception in juvenile and adult female zebra finches. They received lentiviral knockdowns of FoxP1 in one of two brain areas involved in auditory stimulus processing, HVC (proper name) or CMM (caudomedial mesopallium). Ninety-six females, distributed over different experimental and control groups were trained to discriminate between two stimulus songs in an operant Go/Nogo paradigm and subsequently tested with an array of stimuli. This made it possible to assess how well they recognized and categorized altered versions of training stimuli and whether localized FoxP1 knockdowns affected the role of different features during discrimination and categorization of song. Although FoxP1 expression was significantly reduced by the knockdowns, neither discrimination of the stimulus songs nor categorization of songs modified in pitch, sequential order of syllables or by reversed playback were affected. Subsequently, we analyzed the full dataset to assess the impact of the different stimulus manipulations for cue weighing in song discrimination. Our findings show that zebra finches rely on multiple parameters for song discrimination, but with relatively more prominent roles for spectral parameters and syllable sequencing as cues for song discrimination.NEW & NOTEWORTHY In humans, mutations of the transcription factor FoxP1 are implicated in speech and language problems. In songbirds, FoxP1 has been linked to male song learning and female preference strength. We found that FoxP1 knockdowns in female HVC and caudomedial mesopallium (CMM) did not alter song discrimination or categorization based on spectral and temporal information. However, this large dataset allowed to validate different cue weights for spectral over temporal information for song recognition.

Effects of Cortical FoxP1 Knockdowns on Learned Song Preference in Female Zebra Finches.

The search for molecular underpinnings of human vocal communication has focused on genes encoding forkhead-box transcription factors, as rare disruptions of FOXP1, FOXP2, and FOXP4 have been linked to disorders involving speech and language deficits. In male songbirds, an animal model for vocal learning, experimentally altered expression levels of these transcription factors impair song production learning. The relative contributions of auditory processing, motor function or auditory-motor integration to the deficits observed after different FoxP manipulations in songbirds are unknown. To examine the potential effects on auditory learning and development, we focused on female zebra finches (Taeniopygia guttata) that do not sing but develop song memories, which can be assayed in operant preference tests. We tested whether the relatively high levels of FoxP1 expression in forebrain areas implicated in female song preference learning are crucial for the development and/or maintenance of this behavior. Juvenile and adult female zebra finches received FoxP1 knockdowns targeted to HVC (proper name) or to the caudomedial mesopallium (CMM). Irrespective of target site and whether the knockdown took place before (juveniles) or after (adults) the sensitive phase for song memorization, all groups preferred their tutor's song. However, adult females with FoxP1 knockdowns targeted at HVC showed weaker motivation to hear song and weaker song preferences than sham-treated controls, while no such differences were observed after knockdowns in CMM or in juveniles. In summary, FoxP1 knockdowns in the cortical song nucleus HVC were not associated with impaired tutor song memory but reduced motivation to actively request tutor songs.

Immunohistochemical characterization of bipolar cells in four distantly related avian species.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCß, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

Adding colour-realistic video images to audio playbacks increases stimulus engagement but does not enhance vocal learning in zebra finches.

Bird song and human speech are learned early in life and for both cases engagement with live social tutors generally leads to better learning outcomes than passive audio-only exposure. Real-world tutor-tutee relations are normally not uni- but multimodal and observations suggest that visual cues related to sound production might enhance vocal learning. We tested this hypothesis by pairing appropriate, colour-realistic, high frame-rate videos of a singing adult male zebra finch tutor with song playbacks and presenting these stimuli to juvenile zebra finches (Taeniopygia guttata). Juveniles exposed to song playbacks combined with video presentation of a singing bird approached the stimulus more often and spent more time close to it than juveniles exposed to audio playback only or audio playback combined with pixelated and time-reversed videos. However, higher engagement with the realistic audio-visual stimuli was not predictive of better song learning. Thus, although multimodality increased stimulus engagement and biologically relevant video content was more salient than colour and movement equivalent videos, the higher engagement with the realistic audio-visual stimuli did not lead to enhanced vocal learning. Whether the lack of three-dimensionality of a video tutor and/or the lack of meaningful social interaction make them less suitable for facilitating song learning than audio-visual exposure to a live tutor remains to be tested.

Dynamic FoxP2 levels in male zebra finches are linked to morphology of adult-born Area X medium spiny neurons.

The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits. FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2high MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2high MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2high MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2low MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong.

Differential Song Deficits after Lentivirus-Mediated Knockdown of FoxP1, FoxP2, or FoxP4 in Area X of Juvenile Zebra Finches.

Mutations in the transcription factors FOXP1 and FOXP2 are associated with speech impairments. FOXP1 is additionally linked to cognitive deficits, as is FOXP4. These FoxP proteins are highly conserved in vertebrates and expressed in comparable brain regions, including the striatum. In male zebra finches, experimental manipulation of FoxP2 in Area X, a striatal song nucleus essential for vocal production learning, affects song development, adult song production, dendritic spine density, and dopamine-regulated synaptic transmission of striatal neurons. We previously showed that, in the majority of Area X neurons FoxP1, FoxP2, and FoxP4 are coexpressed, can dimerize and multimerize with each other and differentially regulate the expression of target genes. These findings raise the possibility that FoxP1, FoxP2, and FoxP4 (FoxP1/2/4) affect neural function differently and in turn vocal learning. To address this directly, we downregulated FoxP1 or FoxP4 in Area X of juvenile zebra finches and compared the resulting song phenotypes with the previously described inaccurate and incomplete song learning after FoxP2 knockdown. We found that experimental downregulation of FoxP1 and FoxP4 led to impaired song learning with partly similar features as those reported for FoxP2 knockdowns. However, there were also specific differences between the groups, leading us to suggest that specific features of the song are differentially impacted by developmental manipulations of FoxP1/2/4 expression in Area X.SIGNIFICANCE STATEMENT We compared the effects of experimentally reduced expression of the transcription factors FoxP1, FoxP2, and FoxP4 in a striatal song nucleus, Area X, on vocal production learning in juvenile male zebra finches. We show, for the first time, that these temporally and spatially precise manipulations of the three FoxPs affect spectral and temporal song features differentially. This is important because it raises the possibility that the different FoxPs control different aspects of vocal learning through combinatorial gene expression or by acting in different microcircuits within Area X. These results are consistent with the deleterious effects of human FOXP1 and FOXP2 mutations on speech and language and add FOXP4 as a possible candidate gene for vocal disorders.

General isochronous rhythm in echolocation calls and social vocalizations of the bat Saccopteryx bilineata.

Rhythm is an essential component of human speech and music but very little is known about its evolutionary origin and its distribution in animal vocalizations. We found a regular rhythm in three multisyllabic vocalization types (echolocation call sequences, male territorial songs and pup isolation calls) of the neotropical bat Saccopteryx bilineata. The intervals between element onsets were used to fit the rhythm for each individual. For echolocation call sequences, we expected rhythm frequencies around 6-24 Hz, corresponding to the wingbeat in S. bilineata which is strongly coupled to echolocation calls during flight. Surprisingly, we found rhythm frequencies between 6 and 24 Hz not only for echolocation sequences but also for social vocalizations, e.g. male territorial songs and pup isolation calls, which were emitted while bats were stationary. Fourier analysis of element onsets confirmed an isochronous rhythm across individuals and vocalization types. We speculate that attentional tuning to the rhythms of echolocation calls on the receivers' side might make the production of equally steady rhythmic social vocalizations beneficial.

Identification of the neurotransmitter profile of AmFoxP expressing neurons in the honeybee brain using double-label in situ hybridization.

BACKGROUND: FoxP transcription factors play crucial roles for the development and function of vertebrate brains. In humans the neurally expressed FOXPs, FOXP1, FOXP2, and FOXP4 are implicated in cognition, including language. Neural FoxP expression is specific to particular brain regions but FoxP1, FoxP2 and FoxP4 are not limited to a particular neuron or neurotransmitter type. Motor- or sensory activity can regulate FoxP2 expression, e.g. in the striatal nucleus Area X of songbirds and in the auditory thalamus of mice. The DNA-binding domain of FoxP proteins is highly conserved within metazoa, raising the possibility that cellular functions were preserved across deep evolutionary time. We have previously shown in bee brains that FoxP is expressed in eleven specific neuron populations, seven tightly packed clusters and four loosely arranged groups. RESULTS: The present study examined the co-expression of honeybee FoxP (AmFoxP) with markers for glutamatergic, GABAergic, cholinergic and monoaminergic transmission. We found that AmFoxP could co-occur with any one of those markers. Interestingly, AmFoxP clusters and AmFoxP groups differed with respect to homogeneity of marker co-expression; within a cluster, all neurons co-expressed the same neurotransmitter marker, within a group co-expression varied. We also assessed qualitatively whether age or housing conditions providing different sensory and motor experiences affected the AmFoxP neuron populations, but found no differences. CONCLUSIONS: Based on the neurotransmitter homogeneity we conclude that AmFoxP neurons within the clusters might have a common projection and function whereas the AmFoxP groups are more diverse and could be further sub-divided. The obtained information about the neurotransmitters co-expressed in the AmFoxP neuron populations facilitated the search of similar neurons described in the literature. These comparisons revealed e.g. a possible function of AmFoxP neurons in the central complex. Our findings provide opportunities to focus future functional studies on invertebrate FoxP expressing neurons. In a broader context, our data will contribute to the ongoing efforts to discern in which cases relationships between molecular and phenotypic signatures are linked evolutionary.

FoxP in bees: A comparative study on the developmental and adult expression pattern in three bee species considering isoforms and circuitry.

Mutations in the transcription factors FOXP1, FOXP2, and FOXP4 affect human cognition, including language. The FoxP gene locus is evolutionarily ancient and highly conserved in its DNA-binding domain. In Drosophila melanogaster FoxP has been implicated in courtship behavior, decision making, and specific types of motor-learning. Because honeybees (Apis mellifera, Am) excel at navigation and symbolic dance communication, they are a particularly suitable insect species to investigate a potential link between neural FoxP expression and cognition. We characterized two AmFoxP isoforms and mapped their expression in the brain during development and in adult foragers. Using a custom-made antiserum and in situ hybridization, we describe 11 AmFoxP expressing neuron populations. FoxP was expressed in equivalent patterns in two other representatives of Apidae; a closely related dwarf bee and a bumblebee species. Neural tracing revealed that the largest FoxP expressing neuron cluster in honeybees projects into a posterior tract that connects the optic lobe to the posterior lateral protocerebrum, predicting a function in visual processing. Our data provide an entry point for future experiments assessing the function of FoxP in eusocial Hymenoptera.

Fundamental constraints in synchronous muscle limit superfast motor control in vertebrates.

Superfast muscles (SFMs) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz, enabling precise motor execution at the millisecond time scale. SFM phenotypes have been discovered in most major vertebrate lineages, but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed, and if SFMs arose once, or from independent evolutionary events. Here, we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct. Furthermore, we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times. Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish, birds, and mammals, and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle. Consequentially, these constraints set a fundamental limit to the maximum speed of fine motor control.

FoxP expression identifies a Kenyon cell subtype in the honeybee mushroom bodies linking them to fruit fly αßc neurons.

The arthropod mushroom bodies (MB) are a higher order sensory integration centre. In insects, they play a central role in associative olfactory learning and memory. In Drosophila melanogaster (Dm), the highly ordered connectivity of heterogeneous MB neuron populations has been mapped using sophisticated molecular genetic and anatomical techniques. The MB-core subpopulation was recently shown to express the transcription factor FoxP with relevance for decision-making. Here, we report the development and adult distribution of a FoxP-expressing neuron population in the MB of honeybees (Apis mellifera, Am) using in situ hybridisation and a custom-made antiserum. We found the same expression pattern in adult bumblebees (Bombus terrestris, Bt). We also designed a new Dm transgenic line that reports FoxP transcriptional activity in the MB-core region, clarifying previously conflicting data of two other reporter lines. Considering developmental, anatomical and molecular similarities, our data are consistent with the concept of deep homology of FoxP expression in neuron populations coding reinforcement-based learning and habit formation.

Maturation, Behavioral Activation, and Connectivity of Adult-Born Medium Spiny Neurons in a Striatal Song Nucleus.

Neurogenesis continues in the adult songbird brain. Many telencephalic song control regions incorporate new neurons into their existing circuits in adulthood. One song nucleus that receives many new neurons is Area X. Because this striatal region is crucial for song learning and song maintenance the recruitment of new neurons into Area X could influence these processes. As an entry point into addressing this possibility, we investigated the maturation and connectivity within the song circuit and behavioral activation of newly generated Area X neurons. Using BrdU birth dating and virally mediated GFP expression we followed adult-generated neurons from their place of birth in the ventricle to their place of incorporation into Area X. We show that newborn neurons receive glutamatergic input from pallial/cortical song nuclei. Additionally, backfills revealed that the new neurons connect to pallidal-like projection neurons that innervate the thalamus. Using in situ hybridization, we found that new neurons express the mRNA for D1- and D2-type dopamine receptors. Employing DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) and EGR-1 (early growth response protein 1) as markers for neural maturation and activation, we established that at 42 days after labeling approximately 80% of new neurons were mature medium spiny neurons (MSNs) and could be activated by singing behavior. Finally, we compared the MSN density in Area X of birds up to seven years of age and found a significant increase with age, indicating that new neurons are constantly added to the nucleus. In summary, we provide evidence that newborn MSNs in Area X constantly functionally integrate into the circuit and are thus likely to play a role in the maintenance and regulation of adult song.

Protein-Protein Interaction Among the FoxP Family Members and their Regulation of Two Target Genes, VLDLR and CNTNAP2 in the Zebra Finch Song System.

The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP2 contributes to song learning and production in birds. In human cell lines, transcriptional activity of FOXP2 requires homo-dimerization or dimerization with paralogs FOXP1 or FOXP4. Whether FoxP dimerization occurs in the brain is unknown. We recently showed that FoxP1, FoxP2 and FoxP4 (FoxP1/2/4) proteins are co-expressed in neurons of Area X, a song control region in zebra finches. We now report on dimer- and oligomerization of zebra finch FoxPs and how this affects transcription. In cell lines and in the brain we identify homo- and hetero-dimers, and an oligomer composed of FoxP1/2/4. We further show that FoxP1/2 but not FoxP4 bind to the regulatory region of the target gene Contactin-associated protein-like 2 (CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.

Genoarchitecture of the extended amygdala in zebra finch, and expression of FoxP2 in cell corridors of different genetic profile.

We used a battery of genes encoding transcription factors (Pax6, Islet1, Nkx2.1, Lhx6, Lhx5, Lhx9, FoxP2) and neuropeptides to study the extended amygdala in developing zebra finches. We identified different components of the central extended amygdala comparable to those found in mice and chickens, including the intercalated amygdalar cells, the central amygdala, and the lateral bed nucleus of the stria terminalis. Many cells likely originate in the dorsal striatal domain, ventral striatal domain, or the pallidal domain, as is the case in mice and chickens. Moreover, a cell subpopulation of the central extended amygdala appears to originate in the prethalamic eminence. As a general principle, these different cells with specific genetic profiles and embryonic origin form separate or partially intermingled cell corridors along the extended amygdala, which may be involved in different functional pathways. In addition, we identified the medial amygdala of the zebra finch. Like in the chickens and mice, it is located in the subpallium and is rich in cells of pallido-preoptic origin, containing minor subpopulations of immigrant cells from the ventral pallium, alar hypothalamus and prethalamic eminence. We also proposed that the medial bed nucleus of the stria terminalis is composed of several parallel cell corridors with different genetic profile and embryonic origin: preoptic, pallidal, hypothalamic, and prethalamic. Several of these cell corridors with distinct origin express FoxP2, a transcription factor implicated in synaptic plasticity. Our results pave the way for studies using zebra finches to understand the neural basis of social behavior, in which the extended amygdala is involved.

Temporal regularity increases with repertoire complexity in the Australian pied butcherbird's song.

Music maintains a characteristic balance between repetition and novelty. Here, we report a similar balance in singing performances of free-living Australian pied butcherbirds. Their songs include many phrase types. The more phrase types in a bird's repertoire, the more diverse the singing performance can be. However, without sufficient temporal organization, avian listeners may find diverse singing performances difficult to perceive and memorize. We tested for a correlation between the complexity of song repertoire and the temporal regularity of singing performance. We found that different phrase types often share motifs (notes or stereotyped groups of notes). These shared motifs reappeared in strikingly regular temporal intervals across different phrase types, over hundreds of phrases produced without interruption by each bird. We developed a statistical estimate to quantify the degree to which phrase transition structure is optimized for maximizing the regularity of shared motifs. We found that transition probabilities between phrase types tend to maximize regularity in the repetition of shared motifs, but only in birds of high repertoire complexity. Conversely, in birds of low repertoire complexity, shared motifs were produced with less regularity. The strong correlation between repertoire complexity and motif regularity suggests that birds possess a mechanism that regulates the temporal placement of shared motifs in a manner that takes repertoire complexity into account. We discuss alternative musical, mechanistic and ecological explanations to this effect.

"Bird Song Metronomics": Isochronous Organization of Zebra Finch Song Rhythm.

The human capacity for speech and vocal music depends on vocal imitation. Songbirds, in contrast to non-human primates, share this vocal production learning with humans. The process through which birds and humans learn many of their vocalizations as well as the underlying neural system exhibit a number of striking parallels and have been widely researched. In contrast, rhythm, a key feature of language, and music, has received surprisingly little attention in songbirds. Investigating temporal periodicity in bird song has the potential to inform the relationship between neural mechanisms and behavioral output and can also provide insight into the biology and evolution of musicality. Here we present a method to analyze birdsong for an underlying rhythmic regularity. Using the intervals from one note onset to the next as input, we found for each bird an isochronous sequence of time stamps, a "signal-derived pulse," or pulse(S), of which a subset aligned with all note onsets of the bird's song. Fourier analysis corroborated these results. To determine whether this finding was just a byproduct of the duration of notes and intervals typical for zebra finches but not dependent on the individual duration of elements and the sequence in which they are sung, we compared natural songs to models of artificial songs. Note onsets of natural song deviated from the pulse(S) significantly less than those of artificial songs with randomized note and gap durations. Thus, male zebra finch song has the regularity required for a listener to extract a perceived pulse (pulse(P)), as yet untested. Strikingly, in our study, pulses(S) that best fit note onsets often also coincided with the transitions between sub-note elements within complex notes, corresponding to neuromuscular gestures. Gesture durations often equaled one or more pulse(S) periods. This suggests that gesture duration constitutes the basic element of the temporal hierarchy of zebra finch song rhythm, an interesting parallel to the hierarchically structured components of regular rhythms in human music.

What Pinnipeds Have to Say about Human Speech, Music, and the Evolution of Rhythm.

Research on the evolution of human speech and music benefits from hypotheses and data generated in a number of disciplines. The purpose of this article is to illustrate the high relevance of pinniped research for the study of speech, musical rhythm, and their origins, bridging and complementing current research on primates and birds. We briefly discuss speech, vocal learning, and rhythm from an evolutionary and comparative perspective. We review the current state of the art on pinniped communication and behavior relevant to the evolution of human speech and music, showing interesting parallels to hypotheses on rhythmic behavior in early hominids. We suggest future research directions in terms of species to test and empirical data needed.

FoxP2 directly regulates the reelin receptor VLDLR developmentally and by singing.

Mutations of the transcription factor FOXP2 cause a severe speech and language disorder. In songbirds, FoxP2 is expressed in the medium spiny neurons (MSNs) of the avian basal ganglia song nucleus, Area X, which is crucial for song learning and adult song performance. Experimental downregulation of FoxP2 in Area X affects spine formation, prevents neuronal plasticity induced by social context and impairs song learning. Direct target genes of FoxP2 relevant for song learning and song production are unknown. Here we show that a lentivirally mediated FoxP2 knockdown in Area X of zebra finches downregulates the expression of VLDLR, one of the two reelin receptors. Zebra finch FoxP2 binds to the promoter of VLDLR and activates it, establishing VLDLR as a direct FoxP2 target. Consistent with these findings, VLDLR expression is co-regulated with FoxP2 as a consequence of adult singing and during song learning. We also demonstrate that knockdown of FoxP2 affects glutamatergic transmission at the corticostriatal MSN synapse. These data raise the possibility that the regulatory relationship between FoxP2 and VLDLR guides structural plasticity towards the subset of FoxP2-positive MSNs in an activity dependent manner via the reelin pathway.

Young, active and well-connected: adult-born neurons in the zebra finch are activated during singing.

Neuronal replacement in the pallial song control nucleus HVC of adult zebra finches constitutes an interesting case of homeostatic plasticity; in spite of continuous addition and attrition of neurons in ensembles that code song elements, adult song remains remarkably invariant. New neurons migrate into HVC and later synapse with their target, arcopallial song nucleus RA (HVCRA). New HVCRA neurons respond to auditory stimuli (in anaesthetised animals), but whether and when they become functionally active during singing is unknown. We studied this, using 5-bromo-2'-deoxyuridine to birth-date neurons, combined with immunohistochemical detection of immediate-early gene (IEG) expression and retrograde tracer injections into RA to track connectivity. Interestingly, singing was followed by IEG expression in a substantial fraction of new neurons that were not retrogradely labelled from RA, suggesting a possible role in HVC-intrinsic network function. As new HVC neurons matured, the proportion of HVCRA neurons that expressed IEGs after singing increased significantly. Since it was previously shown that singing induces IEG expression in HVC also in deaf birds and that hearing song does not induce IEG expression in HVC, our data provide the first direct evidence that new HVC neurons are engaged in song motor behaviour.

Early developmental stress negatively affects neuronal recruitment to avian song system nucleus HVC.

Adverse environmental conditions can impact the life history trajectory of animals. Adaptive responses enable individuals to cope with unfavorable conditions, but altered metabolism and resource allocation can bear long-term costs. In songbirds, early developmental stress can cause lifelong changes in learned song, a culturally transmitted trait, and nestlings experiencing developmental stress develop smaller song control nucleus HVCs. We investigated whether nutrition-related developmental stress impacts neurogenesis in HVC, which may explain how poor nutrition leads to smaller HVC volume. We provided different quality diets (LOW and HIGH) by varying the husks-to-seeds ratio to zebra finch families for the first 35 days after the young hatched (PHD). At PHD14-18 and again at nutritional independence (PHD35), juveniles were injected with different cell division markers. To monitor growth, we took body measures at PHD10, 17, and 35. At PHD35 the number of newly recruited neurons in HVC and the rate of proliferation in the adjacent ventricular zone (VZ) were counted. Males raised on the LOW diet for their first weeks of life had significantly fewer new neurons in HVC than males raised on the HIGH diet. At the time when these new HVC neurons were born and labeled in the VZ (PHD17) the birds exposed to the LOW diet had significantly lower body mass. At PHD35 body mass or neuronal proliferation no longer differed. Our study shows that even transitory developmental stress can have negative consequences on the cellular processes underlying the development of neural circuits.