Notch1 is involved in cell proliferation and neuronal differentiation in the HVC of zebra finch (Taeniopygia guttata).

Significant sex differences are found in songbirds' song control nuclei and their controlled song behaviors. To elucidate the underlying mechanisms, we explored the role of Notch1 during the development of the high vocal centre (HVC) and song learning in zebra finch. Our study first found that Notch1 positive cells were distributed in HVC with female-biased densities at posthatching day (PHD) 15, but male-biased at PHD 45 and adult. There were about 60 putative oestrogen-responsive elements within 2.5 kb upstream of Notch1, and Notch1 mRNA in the explants that contained the developing male HVC was significantly increased after estrogen addition into the cultured medium for 48 h. After injecting Notch1-interfering lentivirus into the male or female HVC at PHD 15, cell proliferation was significantly promoted in the ventricle zone overlying the HVC at PHD 23. In addition, neuronal differentiation towards Hu+ /BrdU+ at PHD 31, mature neurons (NeuN+/BrdU+) including those projecting to RA in HVC and the sizes of HVC and RA at adult increased significantly after Notch1-interfering lentiviruses were injected into the male HVC at PHD 15. However, the above measurements decreased, following the injection of the lentiviruses expressing Notch intracellular domain (NICD). Finally, the repeat numbers of syllables 'b' or 'c' of learned songs changed after the injection of Notch1-interfering or NICD-expressing lentiviruses into the HVC at PHD15. Our study suggests that Notch1 is related to the development of HVC and song learning in the zebra finch.

The effects of Wnt, BMP, and Notch signaling pathways on cell proliferation and neural differentiation in a song control nucleus (HVC) of Lonchura striata.

There is obvious sexual dimorphism in the song control system of songbirds. In the higher vocal center (HVC), cell proliferation and neuronal differentiation contribute to the net addition of neurons. However, the mechanism underlying these changes is unclear. Given that Wnt, Bmp, and Notch pathways are involved in cell proliferation and neuronal differentiation, no reports are available to study the role of the three pathways in the song control system. To address the issue, we studied cell proliferation in the ventricle zone overlying the developing HVC and neural differentiation within the HVC of Bengalese finches (Lonchura striata) at posthatching day 15 when HVC progenitor cells are generated on a large scale and differentiate into neurons, after Wnt and Bmp pathways were activated by using a pharmacological agonist (LiCl) or Bmp4, respectively, and the Notch pathway was inhibited by an inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), DAPT). The results indicated that both cell proliferation and neural differentiation toward HVC neurons increased significantly after activation of the Wnt signaling pathway or inhibition of the Notch signaling pathway. Although cell proliferation was increased, neural differentiation was inhibited after treatment with Bmp4. There was obvious synergetic enhancement in the number of proliferating cells after the coregulation of two or three signaling pathways. In addition, synergetic enhancement was also found in the Wnt and Notch pathways in neural differentiation toward neurons within HVC. These results suggest that the three signaling pathways are involved in cell proliferation and neural differentiation of HVC.

DARPP-32 distinguishes a subset of adult-born neurons in zebra finch HVC.

Adult male zebra finches (Taeniopygia guttata) continually incorporate adult-born neurons into HVC, a telencephalic brain region necessary for the production of learned song. These neurons express activity-dependent immediate early genes (e.g., zenk and c-fos) following song production, suggesting that these neurons are active during song production. Half of these adult-born HVC neurons (HVC NNs) can be backfilled from the robust nucleus of the arcopallium (RA) and are a part of the vocal motor pathway underlying learned song production, but the other half do not backfill from RA, and they remain to be characterized. Here, we used cell birth-dating, retrograde tract tracing, and immunofluorescence to demonstrate that half of all HVC NNs express the phosphoprotein DARPP-32, a protein associated with dopamine receptor expression. We also demonstrate that DARPP-32+ HVC NNs are contacted by tyrosine hydroxylase immunoreactive fibers, suggesting that they receive catecholaminergic input, have transiently larger nuclei than DARPP-32-neg HVC NNs, and do not backfill from RA. Taken together, these findings help characterize a group of HVC NNs that have no apparent projections to RA and so far have eluded positive identification other than HVC NN status.

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control.

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navß3 to Navß4. Dynamic clamping and dialysis of Navß4's C-terminal peptide into juvenile RA neurons provide evidence that Navß4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Expression of FoxP2 in the basal ganglia regulates vocal motor sequences in the adult songbird.

Disruption of the transcription factor FoxP2, which is enriched in the basal ganglia, impairs vocal development in humans and songbirds. The basal ganglia are important for the selection and sequencing of motor actions, but the circuit mechanisms governing accurate sequencing of learned vocalizations are unknown. Here, we show that expression of FoxP2 in the basal ganglia is vital for the fluent initiation and termination of birdsong, as well as the maintenance of song syllable sequencing in adulthood. Knockdown of FoxP2 imbalances dopamine receptor expression across striatal direct-like and indirect-like pathways, suggesting a role of dopaminergic signaling in regulating vocal motor sequencing. Confirming this prediction, we show that phasic dopamine activation, and not inhibition, during singing drives repetition of song syllables, thus also impairing fluent initiation and termination of birdsong. These findings demonstrate discrete circuit origins for the dysfluent repetition of vocal elements in songbirds, with implications for speech disorders.

Intrinsic plasticity and birdsong learning.

Although information processing and storage in the brain is thought to be primarily orchestrated by synaptic plasticity, other neural mechanisms such as intrinsic plasticity are available. While a number of recent studies have described the plasticity of intrinsic excitability in several types of neurons, the significance of non-synaptic mechanisms in memory and learning remains elusive. After reviewing plasticity of intrinsic excitation in relation to learning and homeostatic mechanisms, we focus on the intrinsic properties of a class of basal-ganglia projecting song system neurons in zebra finch, how these related to each bird's unique learned song, how these properties change over development, and how they are maintained dynamically to rapidly change in response to auditory feedback perturbations. We place these results in the broader theme of learning and changes in intrinsic properties, emphasizing the computational implications of this form of plasticity, which are distinct from synaptic plasticity. The results suggest that exploring reciprocal interactions between intrinsic and network properties will be a fruitful avenue for understanding mechanisms of birdsong learning.

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.

Cannabidiol improves vocal learning-dependent recovery from, and reduces magnitude of deficits following, damage to a cortical-like brain region in a songbird pre-clinical animal model.

Cannabidiol (CBD), a non-euphorigenic compound derived from Cannabis, shows promise for improving recovery following cerebral ischemia and has recently been shown effective for the treatment of childhood seizures caused by Dravet and Lennox-Gastaut syndromes. Given evidence for activity to mitigate effects of CNS insult and dysfunction, we considered the possibility that CBD may also protect and improve functional recovery of a complex learned behavior. To test this hypothesis, we have applied a songbird, the adult male zebra finch, as a novel pre-clinical animal model. Their learned vocalizations were temporarily disrupted with bilateral microlesions of HVC (used as a proper name) a pre-vocal motor cortical-like brain region that drives song. These microlesions destroy about 10% of HVC, and temporarily impair song production, syntax and phonology for about seven days. Recovery requires sensorimotor learning as it depends upon auditory feedback. Four CBD doses (0, 1, 10 and 100 mg/kg) within three surgery conditions (microlesion, no-microlesion, sham-microlesion) were evaluated (n = 5-6). Birds were recorded over 20 days: three baseline; six pre-microlesion drug treatment days and; 11 post-microlesion treatment and recovery days. Results indicate 10 and 100 mg/kg CBD effectively reduced the time required to recover vocal phonology and syntax. In the case of phonology, the magnitude of microlesion-related disruptions were also reduced. These results suggest CBD holds promise to improve functional recovery of complex learned behaviors following brain injury, and represent establishment of an important new animal model to screen drugs for efficacy to improve vocal recovery.

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.

Excitatory and inhibitory synapse reorganization immediately after critical sensory experience in a vocal learner.

Excitatory and inhibitory synapses are the brain's most abundant synapse types. However, little is known about their formation during critical periods of motor skill learning, when sensory experience defines a motor target that animals strive to imitate. In songbirds, we find that exposure to tutor song leads to elimination of excitatory synapses in HVC (used here as a proper name), a key song generating brain area. A similar pruning is associated with song maturation, because juvenile birds have fewer excitatory synapses, the better their song imitations. In contrast, tutoring is associated with rapid insertion of inhibitory synapses, but the tutoring-induced structural imbalance between excitation and inhibition is eliminated during subsequent song maturation. Our work suggests that sensory exposure triggers the developmental onset of goal-specific motor circuits by increasing the relative strength of inhibition and it suggests a synapse-elimination model of song memorization.

Pre-Bout Neural Activity Changes in Premotor Nucleus HVC Correlate with Successful Initiation of Learned Song Sequence.

Preparatory activity, characterized by gradual, longer timescale changes in neural activity, is present in a number of different brain areas before the onset of simple movements and is believed to be important for movement initiation. However, relatively little is known about such activity before initiation of naturally learned movement sequences. The song of an adult male zebra finch is a well studied example of a naturally learned movement sequence and previous studies have shown robust premotor activity immediately before song. Here, I characterize longer timescale changes in neural activity in adult male zebra finch premotor nucleus HVC before onset of song bouts. I show that interneurons and a subset of basal-ganglia-projecting neurons change their activity several hundred milliseconds before song bout onset. Interneurons increased their activity, whereas basal-ganglia-projecting neurons either increased or decreased their activity. Such changes in neural activity were larger, started earlier, and were more common specifically before song bouts that began with the short, repetitive, introductory notes (INs) characteristic of zebra finch song bouts. Further, stronger and earlier changes were also correlated with successful song sequence initiation. Finally, a small fraction of basal-ganglia-projecting neurons that increased their activity before song bout onset did not have song or IN-related activity, suggesting a specialized preparatory role for such neurons. Overall, these data suggest that pre-bout activity in HVC represents preparatory activity important for initiation of a naturally learned movement sequence.SIGNIFICANCE STATEMENT Changes in neuronal activity well before the onset of simple movements are thought to be important for movement initiation. However, a number of animal movements consist of sequences of simple movements and relatively little is known about neuronal activity before such movement sequences. Using adult zebra finch song, a well studied example of a movement sequence, I show here that neurons in premotor nucleus HVC change their activity hundreds of milliseconds before song bout onset. In most neurons, the presence of such changes correlated with successful song sequence initiation. My results show the presence of preparatory neural activity in HVC and suggest a role for HVC in sequence initiation in addition to its established role in song sequence timing.

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.

Regulation of glycosaminoglycan biogenesis is critical for sensitive-period-dependent vocal ontogeny.

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical-period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside-mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical-period-dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401-1412, 2017.

Neuronal Intrinsic Physiology Changes During Development of a Learned Behavior.

Juvenile male zebra finches learn their songs over distinct auditory and sensorimotor stages, the former requiring exposure to an adult tutor song pattern. The cortical premotor nucleus HVC (acronym is name) plays a necessary role in both learning stages, as well as the production of adult song. Consistent with neural network models where synaptic plasticity mediates developmental forms of learning, exposure to tutor song drives changes in the turnover, density, and morphology of HVC synapses during vocal development. A network's output, however, is also influenced by the intrinsic properties (e.g., ion channels) of the component neurons, which could change over development. Here, we use patch clamp recordings to show cell-type-specific changes in the intrinsic physiology of HVC projection neurons as a function of vocal development. Developmental changes in HVC neurons that project to the basal ganglia include an increased voltage sag response to hyperpolarizing currents and an increased rebound depolarization following hyperpolarization. Developmental changes in HVC neurons that project to vocal-motor cortex include a decreased resting membrane potential and an increased spike amplitude. HVC interneurons, however, show a relatively stable range of intrinsic features across vocal development. We used mathematical models to deduce possible changes in ionic currents that underlie the physiological changes and to show that the magnitude of the observed changes could alter HVC circuit function. The results demonstrate developmental plasticity in the intrinsic physiology of HVC projection neurons and suggest that intrinsic plasticity may have a role in the process of song learning.

[Effects of androgens on the long-term depression of HVC-RA pathway in adult male zebra finches].

Androgens can affect the singing behavior via regulating the song control system. In the present study, the effect of androgen on the synaptic plasticity of high vocal center (HVC)-robust nucleus of the arcopallium (RA) pathway was investigated through electrophysiological recording in vivo. We divided the adult male zebra finches into control, castration and castration plus testosterone implantation groups. The changes of long-term depression (LTD) and the paired-pulse facilitation in HVC-RA pathway induced by high-frequency (400 Hz, 2 s) stimulation of HVC were recorded, respectively. The results showed that high-frequency stimulation could effectively induce LTD in control group, but only evoke short-term depression in the castration group. In castration plus testosterone implantation group, LTD was restored. The paired-pulse facilitation was not obvious in the castration group, whereas it was significantly improved in the control and castration plus testosterone implantation groups. These results suggest that androgens may maintain the stability of song by influencing the level of LTD in HVC-RA pathway in adult male zebra finches, and androgens can affect the short-term synaptic plasticity of HVC-RA pathway.

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.

Remodeling of Dendritic Spines in the Avian Vocal Motor Cortex Following Deafening Depends on the Basal Ganglia Circuit.

Deafening elicits a deterioration of learned vocalization, in both humans and songbirds. In songbirds, learned vocal plasticity has been shown to depend on the basal ganglia-cortical circuit, but the underlying cellular basis remains to be clarified. Using confocal imaging and electron microscopy, we examined the effect of deafening on dendritic spines in avian vocal motor cortex, the robust nucleus of the arcopallium (RA), and investigated the role of the basal ganglia circuit in motor cortex plasticity. We found rapid structural changes to RA dendritic spines in response to hearing loss, accompanied by learned song degradation. In particular, the morphological characters of RA spine synaptic contacts between 2 major pathways were altered differently. However, experimental disruption of the basal ganglia circuit, through lesions in song-specialized basal ganglia nucleus Area X, largely prevented both the observed changes to RA dendritic spines and the song deterioration after hearing loss. Our results provide cellular evidence to highlight a key role of the basal ganglia circuit in the motor cortical plasticity that underlies learned vocal plasticity.

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.

A Distributed Recurrent Network Contributes to Temporally Precise Vocalizations.

How do forebrain and brainstem circuits interact to produce temporally precise and reproducible behaviors? Birdsong is an elaborate, temporally precise, and stereotyped vocal behavior controlled by a network of forebrain and brainstem nuclei. An influential idea is that song premotor neurons in a forebrain nucleus (HVC) form a synaptic chain that dictates song timing in a top-down manner. Here we combine physiological, dynamical, and computational methods to show that song timing is not generated solely by a mechanism localized to HVC but instead is the product of a distributed and recurrent synaptic network spanning the forebrain and brainstem, of which HVC is a component.

HVC contributes toward conspecific contact call responding in male Bengalese finches.

The processes of producing and acquiring birdsong, like human speech, utilize interdependent neural systems for vocal learning and production. In addition to song, these brain areas are undoubtedly used for other affiliative behaviors. Oscine sound production is lateralized because their vocal organ contains two independently controlled sound sources. Therefore, songbirds offer a unique opportunity to study the biological relevance of lateralized behavioral control. Bengalese finches (Lonchura striata domestica) produce different types of sound with each sound source: the left sound generator produces tonal frequencies from 1 to 4 kHz and the right sound source produces the lower frequency (<2 kHz) tonal and broadband sounds. We sought to investigate whether the premotor nucleus HVC contributes toward lateralized auditory processing of conspecific vocalizations. We ablated either the left or the right HVC and tested birds with the callback paradigm using female contact calls that were filtered to accentuate particular frequency ranges. The results show that (a) the acoustic frequency of call stimuli drives different patterns of calling behavior and that (b) both HVC nuclei contribute toward contact call production, but HVC ablation does not alter the number of short calls produced upon hearing a female contact call. These data are consistent with the emerging view that the motor production and auditory processing are linked and suggest that HVC may contribute toward affiliative behaviors by promoting the production of contact call responses.