Intrinsic neuronal properties represent song and error in zebra finch vocal learning.

Neurons regulate their intrinsic physiological properties, which could influence network properties and contribute to behavioral plasticity. Recording from adult zebra finch brain slices we show that within each bird basal ganglia Area X-projecting (HVCX) neurons share similar spike waveform morphology and timing of spike trains, with modeling indicating similar magnitudes of five principal ion currents. These properties vary among birds in lawful relation to acoustic similarity of the birds' songs, with adult sibling pairs (same songs) sharing similar waveforms and spiking characteristics. The properties are maintained dynamically: HVCX within juveniles learning to sing show variable properties, whereas the uniformity rapidly degrades within hours in adults singing while exposed to abnormal (delayed) auditory feedback. Thus, within individual birds the population of current magnitudes covary over the arc of development, while rapidly responding to changes in feedback (in adults). This identifies network interactions with intrinsic properties that affect information storage and processing of learned vocalizations.

Female zebra finches do not sing yet share neural pathways necessary for singing in males.

Adult female zebra finches (Taeniopygia guttata), which do not produce learned songs, have long been thought to possess only vestiges of the forebrain network that supports learned song in males. This view ostensibly explains why females do not sing-many of the neural populations and pathways that make up the male song control network appear rudimentary or even missing in females. For example, classic studies of vocal-premotor cortex (HVC, acronym is name) in male zebra finches identified prominent efferent pathways from HVC to vocal-motor cortex (RA, robust nucleus of the arcopallium) and from HVC to the avian basal ganglia (Area X). In females, by comparison, the efferent targets of HVC were thought to be only partially innervated by HVC axons (RA) or absent (Area X). Here, using a novel visually guided surgical approach to target tracer injections with precision, we mapped the extrinsic connectivity of the adult female HVC. We find that female HVC shows a mostly male-typical pattern of afferent and efferent connectivity, including robust HVC innervation of RA and Area X. As noted by earlier investigators, we find large sex differences in the volume of many regions that control male singing (male > female). However, sex differences in volume were diminished in regions that convey ascending afferent input to HVC. Our findings do not support a vestigial interpretation of the song control network in females. Instead, our findings support the emerging view that the song control network may have an altogether different function in nonsinging females.

Morphological characterization of HVC projection neurons in the zebra finch (Taeniopygia guttata).

Singing behavior in the adult male zebra finch is dependent upon the activity of a cortical region known as HVC (proper name). The vast majority of HVC projection neurons send primary axons to either the downstream premotor nucleus RA (robust nucleus of the arcopallium, or primary motor cortex) or Area X (basal ganglia), which play important roles in song production or song learning, respectively. In addition to these long-range outputs, HVC neurons also send local axon collaterals throughout that nucleus. Despite their implications for a range of circuit models, these local processes have never been completely reconstructed. Here, we use in vivo single-neuron Neurobiotin fills to examine 40 projection neurons across 31 birds with somatic positions distributed across HVC. We show that HVC(RA) and HVC(X) neurons have categorically distinct dendritic fields. Additionally, these cell classes send axon collaterals that are either restricted to a small portion of HVC ("local neurons") or broadly distributed throughout the entire nucleus ("broadcast neurons"). Overall, these processes within HVC offer a structural basis for significant local processing underlying behaviorally relevant population activity.

Orthogonal topography in the parallel input architecture of songbird HVC.

Neural activity within the cortical premotor nucleus HVC (acronym is name) encodes the learned songs of adult male zebra finches (Taeniopygia guttata). HVC activity is driven and/or modulated by a group of five afferent nuclei (the Medial Magnocellular nucleus of the Anterior Nidopallium, MMAN; Nucleus Interface, NIf; nucleus Avalanche, Av; the Robust nucleus of the Arcopallium, RA; the Uvaeform nucleus, Uva). While earlier evidence suggested that HVC receives a uniformly distributed and nontopographic pattern of afferent input, recent evidence suggests this view is incorrect (Basista et al., ). Here, we used a double-labeling strategy (varying both the distance between and the axial orientation of dual tracer injections into HVC) to reveal a massively parallel and in some cases topographic pattern of afferent input. Afferent neurons target only one rostral or caudal location within medial or lateral HVC, and each HVC location receives convergent input from each afferent nucleus in parallel. Quantifying the distributions of single-labeled cells revealed an orthogonal topography in the organization of afferent input from MMAN and NIf, two cortical nuclei necessary for song learning. MMAN input is organized across the lateral-medial axis whereas NIf input is organized across the rostral-caudal axis. To the extent that HVC activity is influenced by afferent input during the learning, perception, or production of song, functional models of HVC activity may need revision to account for the parallel input architecture of HVC, along with the orthogonal input topography of MMAN and NIf.

Exploring sex differences in the adult zebra finch brain: In vivo diffusion tensor imaging and ex vivo super-resolution track density imaging.

Zebra finches are an excellent model to study the process of vocal learning, a complex socially-learned tool of communication that forms the basis of spoken human language. So far, structural investigation of the zebra finch brain has been performed ex vivo using invasive methods such as histology. These methods are highly specific, however, they strongly interfere with performing whole-brain analyses and exclude longitudinal studies aimed at establishing causal correlations between neuroplastic events and specific behavioral performances. Therefore, the aim of the current study was to implement an in vivo Diffusion Tensor Imaging (DTI) protocol sensitive enough to detect structural sex differences in the adult zebra finch brain. Voxel-wise comparison of male and female DTI parameter maps shows clear differences in several components of the song control system (i.e. Area X surroundings, the high vocal center (HVC) and the lateral magnocellular nucleus of the anterior nidopallium (LMAN)), which corroborate previous findings and are in line with the clear behavioral difference as only males sing. Furthermore, to obtain additional insights into the 3-dimensional organization of the zebra finch brain and clarify findings obtained by the in vivo study, ex vivo DTI data of the male and female brain were acquired as well, using a recently established super-resolution reconstruction (SRR) imaging strategy. Interestingly, the SRR-DTI approach led to a marked reduction in acquisition time without interfering with the (spatial and angular) resolution and SNR which enabled to acquire a data set characterized by a 78µm isotropic resolution including 90 diffusion gradient directions within 44h of scanning time. Based on the reconstructed SRR-DTI maps, whole brain probabilistic Track Density Imaging (TDI) was performed for the purpose of super resolved track density imaging, further pushing the resolution up to 40µm isotropic. The DTI and TDI maps realized atlas-quality anatomical maps that enable a clear delineation of most components of the song control and auditory systems. In conclusion, this study paves the way for longitudinal in vivo and high-resolution ex vivo experiments aimed at disentangling neuroplastic events that characterize the critical period for vocal learning in zebra finch ontogeny.

Axial organization of a brain region that sequences a learned pattern of behavior.

Neural activity within HVC (proper name), a premotor nucleus of the songbird telencephalon analogous to premotor cortical regions in mammals, controls the temporal structure of learned song in male zebra finches (Taeniopygia guttata). HVC is composed of a superficially isomorphic neuronal mosaic, implying that song is encoded in a distributed network within HVC. Here, we combined HVC microlesions (10% focal ablation) with singing-driven immediate-early gene (IEG) labeling to explore the network architecture of HVC during singing. Microlesions produce a transient disruption of HVC activity that results in a temporary (≈ 1 week) loss of vocal patterning. Results showed an asymmetrical reduction in the density of IEG-labeled cells 3-5 d after microlesions: swaths of unlabeled cells extended rostrally and/or caudally depending on the position of the HVC microlesion. Labeling returned once birds recovered their songs. Axial swaths of unlabeled cells occurred whether microlesions were located at rostral or caudal poles of HVC, indicating that the localized reduction in IEG labeling could not be attributable solely to transection of afferents that enter HVC rostrally. The asymmetrical pattern of reduced IEG labeling could be explained if synaptic connectivity within HVC is organized preferentially within the rostrocaudal axis. In vivo retrograde tracer injections and in vitro stimulation and recording experiments in horizontal slices of HVC confirmed a rostrocaudal organization of HVC neural connectivity. Our findings suggest that HVC contains an axially organized network architecture that may encode the temporal structure of song.

Male bias in the song control system despite female bias in song rate in streak-backed orioles (Icterus pustulatus).

The song control system is a group of discrete interconnected nuclei found in the brains of all songbirds (suborder Passeri). Previous studies have reported a positive relationship between sex differences in song nucleus volumes and sex differences in song behavior across numerous songbird species, with species exhibiting greater sex differences in behavior also exhibiting greater sex differences in the brain. This body of comparative research, however, has failed to incorporate data from a bird species in which females sing more than males. In this study, we examine song nucleus volumes in both sexes of the streak-backed oriole (Icterus pustulatus), a New World blackbird with a female bias in song rate and similar song complexity between the sexes. Results from this neuroanatomical analysis are contrary to what was to be expected from previous research: despite the female bias in song rate, males have a significantly larger HVC and area X song nucleus volumes. Specifically, male HVC was 75% larger than that of females, and male area X was 64% larger than that of females. There was no significant sex difference in the size of the nucleus robustus arcopallialis. The lack of a positive relationship between song nuclei and singing behavior in these orioles demonstrates that our current understanding of song modulation via the song control system may be overly reliant on basic measures such as total volumes.

Modulation of perineuronal nets and parvalbumin with developmental song learning.

Neural circuits and behavior are shaped during developmental phases of maximal plasticity known as sensitive or critical periods. Neural correlates of sensory critical periods have been identified, but their roles remain unclear. Factors that define critical periods in sensorimotor circuits and behavior are not known. Birdsong learning in the zebra finch occurs during a sensitive period similar to that for human speech. We now show that perineuronal nets, which correlate with sensory critical periods, surround parvalbumin-positive neurons in brain areas that are dedicated to singing. The percentage of both total and parvalbumin-positive neurons with perineuronal nets increased with development. In HVC (this acronym is the proper name), a song area important for sensorimotor integration, the percentage of parvalbumin neurons with perineuronal nets correlated with song maturity. Shifting the vocal critical period with tutor song deprivation decreased the percentage of neurons that were parvalbumin positive and the relative staining intensity of both parvalbumin and a component of perineuronal nets. Developmental song learning shares key characteristics with sensory critical periods, suggesting shared underlying mechanisms.

Rapid interhemispheric switching during vocal production in a songbird.

To generate complex bilateral motor patterns such as those underlying birdsong, neural activity must be highly coordinated across the two cerebral hemispheres. However, it remains largely elusive how this coordination is achieved given that interhemispheric communication between song-control areas in the avian cerebrum is restricted to projections received from bilaterally connecting areas in the mid- and hindbrain. By electrically stimulating cerebral premotor areas in zebra finches, we find that behavioral effectiveness of stimulation rapidly switches between hemispheres. In time intervals in which stimulation in one hemisphere tends to distort songs, stimulation in the other hemisphere is mostly ineffective, revealing an idiosyncratic form of motor dominance that bounces back and forth between hemispheres like a virtual ping-pong ball. The intervals of lateralized effectiveness are broadly distributed and are unrelated to simple spectral and temporal song features. Such interhemispheric switching could be an important dynamical aspect of neural coordination that may have evolved from simpler pattern generator circuits.

Nest of origin predicts adult neuron addition rates in the vocal control system of the zebra finch.

Neurogenesis and neuronal replacement in adulthood represent dramatic forms of plasticity that might serve as a substrate for behavioral flexibility. In songbirds, neurons are continually replaced in HVC (used as a proper name), a pre-motor region necessary for the production of learned vocalizations. There are large individual differences in HVC neuron addition. Some of this variation is probably due to individual differences in adult experience; however, it is also possible that heritability or experience early in development constrains the levels of adult neuron addition. As a step toward addressing the latter two possibilities, we explored the extent to which nest of origin predicts rates of HVC neuron addition in adult male zebra finches. One month after injections of [(3)H]-thymidine to mark dividing cells, neuron addition in HVC was found to co-vary among birds that had been nest mates, even when they were housed in different cages as adults. We also tested whether nest mate co-variation might be due to shared adult auditory experience by measuring neuron addition in nest mate pairs after one member was deafened. There were significant differences in neuron addition between hearing and deaf birds but nest mate relationships persisted. These results suggest that variation in genotype and/or early pre- or postnatal experience can account for a large fraction of adult variation in rates of neuron addition. These results also suggest that a major constraint on neurogenesis and the capacity to adjust rates of neuron addition in response to adult auditory experience is established early in development.

Functional identification of sensory mechanisms required for developmental song learning.

A young male zebra finch (Taeniopygia guttata) learns to sing by copying the vocalizations of an older tutor in a process that parallels human speech acquisition. Brain pathways that control song production are well defined, but little is known about the sites and mechanisms of tutor song memorization. Here we test the hypothesis that molecular signaling in a sensory brain area outside of the song system is required for developmental song learning. Using controlled tutoring and a pharmacological inhibitor, we transiently suppressed the extracellular signal-regulated kinase signaling pathway in a portion of the auditory forebrain specifically during tutor song exposure. On maturation, treated birds produced poor copies of tutor song, whereas controls copied the tutor song effectively. Thus the foundation of normal song learning, the formation of a sensory memory of tutor song, requires a conserved molecular pathway in a brain area that is distinct from the circuit for song motor control.

Testosterone and social context affect singing behavior but not song control region volumes in adult male songbirds in the fall.

Testosterone (T) induces singing behavior and mediates changes in the sizes and neuroanatomical characteristics of brain regions controlling singing behavior (song control regions, SCRs) in songbirds. These effects may require the enzymatic conversion of T into androgenic and estrogenic metabolites by brain tissues and can be modulated by factors such as season and social context. Testosterone administration to adult male House Finches, Carpodacus mexicanus, in the spring increases the size of their SCRs. Here, we used males of this species to investigate effects of T and T metabolism on brain morphology and singing behavior in the fall. Birds received Silastic capsules containing androgens, estrogens, and/or inhibitors of androgenic action or estrogen synthesis to determine effects of these hormones on song rates and SCR volumes. We also manipulated the social environment by changing the number of birds in visual contact with each other. Testosterone treatment stimulated singing behavior in finches held in small, visually isolated groups and exposed to song playbacks. However, administration of T or T metabolites did not increase SCR sizes. The data suggest that photoperiodic condition and social context may modulate the effects of steroids on SCRs and singing behavior.

Hemispheric coordination is necessary for song production in adult birds: implications for a dual role for forebrain nuclei in vocal motor control.

Precise coordination across hemispheres is a critical feature of many complex motor circuits. In the avian song system the robust nucleus of the arcopallium (RA) plays a key role in such coordination. It is simultaneously the major output structure for the descending vocal motor pathway, and it also sends inputs to structures in the brain stem and thalamus that project bilaterally back to the forebrain. Because all birds lack a corpus callosum and the anterior commissure does not interconnect any of the song control nuclei directly, these bottom-up connections form the only pathway that can coordinate activity across hemispheres. In this study, we show that unilateral lesions of RA in adult male zebra finches (Taeniopigia guttata) completely and permanently disrupt the bird's stereotyped song. In contrast, lesions of RA in juvenile birds do not prevent the acquisition of normal song as adults. These results highlight the importance of hemispheric interdependence once the circuit is established but show that one hemisphere is sufficient for complex vocal behavior if this interdependence is prevented during a critical period of development. The ability of birds to sing with a single RA provides the opportunity to test the effect of targeted microlesions in RA without confound of functional compensation from the contralateral RA. We show that microlesions cause significant changes in song temporal structure and implicate RA as playing a major part in the generation of song temporal patterns. These findings implicate a dual role for RA, first as part of the program generator for song and second as part of the circuit that mediates interhemispheric coordination.

LMAN lesions prevent song degradation after deafening without reducing HVC neuron addition.

In some songbirds perturbing auditory feedback can promote changes in song structure well beyond the end of song learning. One factor that may drive vocal change in such deafened birds is the ongoing addition of new vocal-motor neurons into the song system. Without auditory feedback to guide their incorporation, the addition of these new neurons could disrupt the established song pattern. To assess this hypothesis, the authors determined if neuronal recruitment into the vocal motor nucleus HVC is affected by neural signals that influence vocal change in adult deafened birds. Such signals appear to be conveyed via LMAN, a nucleus in the anterior forebrain that is necessary for vocal change after deafening. Here the authors tested whether LMAN lesions might restrict song degradation after deafening by reducing the addition or survival of new HVC neurons that would otherwise corrupt the ongoing song pattern. Using [3H]thymidine autoradiography to identify neurons generated in adult zebra finches, it was shown here that LMAN lesions do not reduce the number or percent of new HVC neurons surviving for either several weeks or months after [3H]thymidine labeling. However, the authors confirmed previous reports that LMAN lesions restrict vocal change after deafening. These data suggest that neurons incorporated into the adult HVC may form behaviorally adaptive connections without requiring auditory feedback, and that any role such neurons may play in promoting vocal change after adult deafening requires anterior forebrain pathway output.

Song repertoire size varies with HVC volume and is indicative of male quality in song sparrows (Melospiza melodia).

Complex birdsong is a classic example of a sexually selected ornamental trait. In many species, females prefer males with large song repertoires, possibly because repertoire size is limited by the size of song control nuclei which reflect developmental success. We investigated whether song repertoire size was indicative of brain area and male quality in song sparrows (Melospiza melodia) by determining if repertoire size was related to the volume of song control nucleus HVC, as well as several morphological, immunological and genetic indices of quality. We found that males with large repertoires had larger HVCs and were in better body condition. They also had lower heterophil to lymphocyte ratios, indicating less physiological stress and a robust immune system as measured by the number of lymphocytes per red blood cell. Song repertoire size also tended to increase with neutral-locus genetic diversity, as assessed by mean d2, but was not related to internal relatedness. Our results suggest several mechanisms that might explain the finding of a recent study that song sparrows with large song repertoires have higher lifetime fitness.

Song and brain development in canaries raised under different conditions of acoustic and social isolation over two years.

Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large normal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a significant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no significant change in repertoire size. Nor did they show any significant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year.

Noradrenergic projections to the song control nucleus area X of the medial striatum in male zebra finches (Taeniopygia guttata).

There is considerable functional evidence implicating norepinephrine in modulating activity in the vocal control circuit of songbirds. However, our knowledge of noradrenergic inputs to the song system is incomplete. In this study, cholera toxin subunit B (CTB) injections into area X revealed projections from the noradrenergic nuclei locus coeruleus and subcoeruleus, and injections of biotinylated dextran amines into these noradrenergic nuclei labeled fibers in area X. The nonreciprocity of this connection was demonstrated by the absence of retrogradely labeled cells in area X following injections of CTB into the locus coeruleus. Additionally, we found novel inputs to area X from the nidopallium and arcopallium, the mesencephalic central gray, and the dorsolateralis anterior (DLL) and posterior (DLP) lateralis in the thalamus. Area X can be clearly distinguished from the surrounding medial striatum based on cytoarchitectural and chemical neuroanatomical criteria. We show here that neuromodulatory inputs to area X however, exhibit a considerable degree of overlap with the surrounding area. This finding suggests that regional specificity in neuromodulator action is most likely afforded by a specialization in receptor density and enzyme distribution rather than projections from the synthesizing nuclei. Our results extend current knowledge about noradrenergic projections to specialized nuclei of the song control circuit and provide neuroanatomical evidence for the functional action of norepinephrine-modulating context-dependent ZENK expression in area X. Furthermore, the novel projections to area X from telencephalic and thalamic areas could be new and interesting nodes in the striatopallidothalamic loop spanning the songbird brain.

Sleep-related spike bursts in HVC are driven by the nucleus interface of the nidopallium.

The function and the origin of replay of motor activity during sleep are currently unknown. Spontaneous activity patterns in the nucleus robustus of the arcopallium (RA) and in HVC (high vocal center) of the sleeping songbird resemble premotor patterns in these areas observed during singing. We test the hypothesis that the nucleus interface of the nidopallium (NIf) has an important role for initiating and shaping these sleep-related activity patterns. In head-fixed, sleeping zebra finches we find that injections of the GABA(A)-agonist muscimol into NIf lead to transient abolishment of premotor-like bursting activity in HVC neurons. Using antidromic activation of NIf neurons by electrical stimulation in HVC, we are able to distinguish a class of HVC-projecting NIf neurons from a second class of NIf neurons. Paired extracellular recordings in NIf and HVC show that NIf neurons provide a strong bursting drive to HVC. In contrast to HVC neurons, whose bursting activity waxes and wanes in burst epochs, individual NIf projection neurons are nearly continuously bursting and tend to burst only once on the timescale of song syllables. Two types of HVC projection neurons-premotor and striatal projecting-respond differently to the NIf drive, in agreement with notions of HVC relaying premotor signals to RA and an anticipatory copy thereof to areas of a basal ganglia pathway.

Developmental modulation of the temporal relationship between brain and behavior.

Humans and songbirds shape learned vocalizations during a sensorimotor sensitive period or "babbling" phase. The brain mechanisms that underlie the shaping of vocalizations by sensory feedback are not known. We examined song behavior and brain activity in zebra finches during singing as they actively shaped their song toward a tutor model. We now show that the temporal relationship of behavior and activity in the premotor area HVC changes with the development of song behavior. During sensorimotor learning, HVC bursting activity both preceded and followed learned vocalizations by hundreds of milliseconds. Correspondingly, the duration of bursts that occurred during ongoing song motif behavior was prolonged in juveniles, as compared with adults, and was inversely correlated with song maturation. Multielectrode single-unit recording in juveniles revealed that single fast-spiking neurons were active both before and after vocalization. These same neurons responded to auditory stimuli. Collectively, these data indicate that a key aspect of sensory critical periods--prolonged bursting--also applies to sensorimotor development. In addition, prolonged motor discharge and sensory input coincide in single neurons of the developing song system, providing the necessary cellular elements for sensorimotor shaping through activity-dependent mechanisms.

The distribution of substance P and met-enkephalin in vocal control nuclei among oscine species and its relation to song complexity.

Substance P (SP) and methionine-enkephalin (ENK) have been reported to appear in song control nuclei of oscine species. However, it remains unknown whether or not SP and ENK location in song control nuclei is correlated with song behavior. To address this issue, the present study first measured two variables for song complexity, i.e., song repertoire sizes, and syllable repertoire sizes in 11 oscine species. Then, we examined the distribution of SP and ENK in four control nuclei, two in the motor pathway, i.e., HVC and the robust nucleus of arcopallium (RA), and the other two in the forebrain pathway, i.e., Area X and the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Finally, we measured the relative amounts of immunoreactivity for SP and ENK in song control nuclei, and tested whether they were correlated with song complexity. Our results showed that: (1) SP and ENK were broadly distributed in the song control nuclei of studied species. However, SP immunohistochemistry was more robust in comparison with ENK, and SP is generally more abundant in the two song learning nuclei than those in the two song producing ones; (2) SP and ENK staining patterns in song control nuclei did not show any obvious phylogenetic relationship among studied oscine species; (3) there was a significant correlation between the relative amounts of immunoreactivity for SP and the song and syllable repertoire sizes. Our results suggest that SP or ENK might be involved in song behavior, such as birdsong learning or memory.