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.

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.

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.

[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.

Interplay of inhibition and excitation shapes a premotor neural sequence.

In the zebra finch, singing behavior is driven by a sequence of bursts within premotor neurons located in the forebrain nucleus HVC (proper name). In addition to these excitatory projection neurons, HVC also contains inhibitory interneurons with a role in premotor patterning that is unclear. Here, we used a range of electrophysiological and behavioral observations to test previously described models suggesting discrete functional roles for inhibitory interneurons in song production. We show that single HVC premotor neuron bursts are sufficient to drive structured activity within the interneuron network because of pervasive and facilitating synaptic connections. We characterize interneuron activity during singing and describe reliable pauses in the firing of those neurons. We then demonstrate that these gaps in inhibition are likely to be necessary for driving normal bursting behavior in HVC premotor neurons and suggest that structured inhibition and excitation may be a general mechanism enabling sequence generation in other circuits.

Independent premotor encoding of the sequence and structure of birdsong in avian cortex.

How the brain coordinates rapid sequences of learned behavior, such as human speech, remains a fundamental problem in neuroscience. Birdsong is a model of such behavior, which is learned and controlled by a neural circuit that spans avian cortex, basal ganglia, and thalamus. The songs of adult male zebra finches (Taeniopygia guttata), produced as rapid sequences of vocal gestures (syllables), are encoded by the cortical premotor region HVC (proper name). While the motor encoding of song within HVC has traditionally been viewed as unitary and distributed, we used an ablation technique to ask whether the sequence and structure of song are processed independently within HVC. Results revealed a functional topography across the medial-lateral axis of HVC. Bilateral ablation of medial HVC induced a positive disruption of song (increase in atypical syllable sequences), whereas bilateral ablation of lateral HVC induced a negative disruption (omission of individual syllables). Bilateral ablation of central HVC either had no effect on song or induced syllable omission, similar to lateral HVC ablation. We then investigated HVC connectivity and found parallel afferent and efferent pathways that transit medial and lateral HVC and converge at vocal motor cortex. In light of recent evidence that syntactic and lexical components of human speech are processed independently by neighboring regions of cortex (Menenti et al., 2012), our demonstration of anatomically distinct pathways that differentially process the sequence and structure of birdsong in parallel suggests that the vertebrate brain relies on a common approach to encode rapid sequences of vocal gestures.

Automatic reconstruction of physiological gestures used in a model of birdsong production.

Highly coordinated learned behaviors are key to understanding neural processes integrating the body and the environment. Birdsong production is a widely studied example of such behavior in which numerous thoracic muscles control respiratory inspiration and expiration: the muscles of the syrinx control syringeal membrane tension, while upper vocal tract morphology controls resonances that modulate the vocal system output. All these muscles have to be coordinated in precise sequences to generate the elaborate vocalizations that characterize an individual's song. Previously we used a low-dimensional description of the biomechanics of birdsong production to investigate the associated neural codes, an approach that complements traditional spectrographic analysis. The prior study used algorithmic yet manual procedures to model singing behavior. In the present work, we present an automatic procedure to extract low-dimensional motor gestures that could predict vocal behavior. We recorded zebra finch songs and generated synthetic copies automatically, using a biomechanical model for the vocal apparatus and vocal tract. This dynamical model described song as a sequence of physiological parameters the birds control during singing. To validate this procedure, we recorded electrophysiological activity of the telencephalic nucleus HVC. HVC neurons were highly selective to the auditory presentation of the bird's own song (BOS) and gave similar selective responses to the automatically generated synthetic model of song (AUTO). Our results demonstrate meaningful dimensionality reduction in terms of physiological parameters that individual birds could actually control. Furthermore, this methodology can be extended to other vocal systems to study fine motor control.

Using temperature to analyse temporal dynamics in the songbird motor pathway.

Many complex behaviours, like speech or music, have a hierarchical organization with structure on many timescales, but it is not known how the brain controls the timing of behavioural sequences, or whether different circuits control different timescales of the behaviour. Here we address these issues by using temperature to manipulate the biophysical dynamics in different regions of the songbird forebrain involved in song production. We find that cooling the premotor nucleus HVC (formerly known as the high vocal centre) slows song speed across all timescales by up to 45 per cent but only slightly alters the acoustic structure, whereas cooling the downstream motor nucleus RA (robust nucleus of the arcopallium) has no observable effect on song timing. Our observations suggest that dynamics within HVC are involved in the control of song timing, perhaps through a chain-like organization. Local manipulation of brain temperature should be broadly applicable to the identification of neural circuitry that controls the timing of behavioural sequences and, more generally, to the study of the origin and role of oscillatory and other forms of brain dynamics in neural systems.

Precise auditory-vocal mirroring in neurons for learned vocal communication.

Brain mechanisms for communication must establish a correspondence between sensory and motor codes used to represent the signal. One idea is that this correspondence is established at the level of single neurons that are active when the individual performs a particular gesture or observes a similar gesture performed by another individual. Although neurons that display a precise auditory-vocal correspondence could facilitate vocal communication, they have yet to be identified. Here we report that a certain class of neurons in the swamp sparrow forebrain displays a precise auditory-vocal correspondence. We show that these neurons respond in a temporally precise fashion to auditory presentation of certain note sequences in this songbird's repertoire and to similar note sequences in other birds' songs. These neurons display nearly identical patterns of activity when the bird sings the same sequence, and disrupting auditory feedback does not alter this singing-related activity, indicating it is motor in nature. Furthermore, these neurons innervate striatal structures important for song learning, raising the possibility that singing-related activity in these cells is compared to auditory feedback to guide vocal learning.

Adult neurogenesis is associated with the maintenance of a stereotyped, learned motor behavior.

Adult neurogenesis is thought to provide neural plasticity used in forming and storing new memories. Here we show a novel relationship between numbers of new neurons and the stability of a previously learned motor pattern. In the adult zebra finch, new projection neurons are added to the nucleus HVC and become part of the motor pathway for producing learned song. However, new song learning occurs only in juveniles and the behavioral impact of adding new neurons to HVC throughout life is unclear. We report that song changes after deafening are inversely correlated with the number of new neurons added to HVC, suggesting that adult neurogenesis in this context may contribute to behavioral stability. More broadly, we propose that new neuron function may depend on the site of integration and can vary as widely as promoting, or restricting, behavioral plasticity.

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.

Afferents from vocal motor and respiratory effectors are recruited during vocal production in juvenile songbirds.

Learned behaviors require coordination of diverse sensory inputs with motivational and motor systems. Although mechanisms underlying vocal learning in songbirds have focused primarily on auditory inputs, it is likely that sensory inputs from vocal effectors also provide essential feedback. We investigated the role of somatosensory and respiratory inputs from vocal effectors of juvenile zebra finches (Taeniopygia guttata) during the stage of sensorimotor integration when they are learning to imitate a previously memorized tutor song. We report that song production induced expression of the immediate early gene product Fos in trigeminal regions that receive hypoglossal afferents from the tongue and syrinx (the main vocal organ). Furthermore, unilateral lesion of hypoglossal afferents greatly diminished singing-induced Fos expression on the side ipsilateral to the lesion, but not on the intact control side. In addition, unilateral lesion of the vagus reduced Fos expression in the ipsilateral nucleus of the solitary tract in singing birds. Lesion of the hypoglossal nerve to the syrinx greatly disrupted vocal behavior, whereas lesion of the hypoglossal nerve to the tongue exerted no obvious disruption and lesions of the vagus caused some alterations to song behavior. These results provide the first functional evidence that somatosensory and respiratory feedback from peripheral effectors is activated during vocal production and conveyed to brainstem regions. Such feedback is likely to play an important role in vocal learning during sensorimotor integration in juvenile birds and in maintaining stereotyped vocal behavior in adults.

Recurrent interactions between the input and output of a songbird cortico-basal ganglia pathway are implicated in vocal sequence variability.

Complex brain functions, such as the capacity to learn and modulate vocal sequences, depend on activity propagation in highly distributed neural networks. To explore the synaptic basis of activity propagation in such networks, we made dual in vivo intracellular recordings in anesthetized zebra finches from the input (nucleus HVC, used here as a proper name) and output [lateral magnocellular nucleus of the anterior nidopallium (LMAN)] neurons of a songbird cortico-basal ganglia (BG) pathway necessary to the learning and modulation of vocal motor sequences. These recordings reveal evidence of bidirectional interactions, rather than only feedforward propagation of activity from HVC to LMAN, as had been previously supposed. A combination of dual and triple recording configurations and pharmacological manipulations was used to map out circuitry by which activity propagates from LMAN to HVC. These experiments indicate that activity travels to HVC through at least two independent ipsilateral pathways, one of which involves fast signaling through a midbrain dopaminergic cell group, reminiscent of recurrent mesocortical loops described in mammals. We then used in vivo pharmacological manipulations to establish that augmented LMAN activity is sufficient to restore high levels of sequence variability in adult birds, suggesting that recurrent interactions through highly distributed forebrain-midbrain pathways can modulate learned vocal sequences.

Electrophysiological characterization and computational models of HVC neurons in the zebra finch.

The nucleus HVC (proper name) within the avian analog of mammal premotor cortex produces stereotyped instructions through the motor pathway leading to precise, learned vocalization by songbirds. Electrophysiological characterization of component HVC neurons is an important requirement in building a model to understand HVC function. The HVC contains three neural populations: neurons that project to the RA (robust nucleus of arcopallium), neurons that project to Area X (of the avian basal ganglia), and interneurons. These three populations are interconnected with specific patterns of excitatory and inhibitory connectivity, and they fire with characteristic patterns both in vivo and in vitro. We performed whole cell current-clamp recordings on HVC neurons within brain slices to examine their intrinsic firing properties and determine which ionic currents are responsible for their characteristic firing patterns. We also developed conductance-based models for the different neurons and calibrated the models using data from our brain slice work. These models were then used to generate predictions about the makeup of the ionic currents that are responsible for the different responses to stimuli. These predictions were then tested and verified in the slice using pharmacological manipulations. The model and the slice work highlight roles of a hyperpolarization-activated inward current (Ih), a low-threshold T-type Ca(2+) current (ICa-T), an A-type K(+) current (IA), a Ca(2+)-activated K(+) current (ISK), and a Na(+)-dependent K(+) current (IKNa) in driving the characteristic neural patterns observed in the three HVC neuronal populations. The result is an improved characterization of the HVC neurons responsible for song production in the songbird.

Directed functional connectivity matures with motor learning in a cortical pattern generator.

Sequential motor skills may be encoded by feedforward networks that consist of groups of neurons that fire in sequence (Abeles 1991; Long et al. 2010). However, there has been no evidence of an anatomic map of activation sequence in motor control circuits, which would be potentially detectable as directed functional connectivity of coactive neuron groups. The proposed pattern generator for birdsong, the HVC (Long and Fee 2008; Vu et al. 1994), contains axons that are preferentially oriented in the rostrocaudal axis (Nottebohm et al. 1982; Stauffer et al. 2012). We used four-tetrode recordings to assess the activity of ensembles of single neurons along the rostrocaudal HVC axis in anesthetized zebra finches. We found an axial, polarized neural network in which sequential activity is directionally organized along the rostrocaudal axis in adult males, who produce a stereotyped song. Principal neurons fired in rostrocaudal order and with interneurons that were rostral to them, suggesting that groups of excitatory neurons fire at the leading edge of travelling waves of inhibition. Consistent with the synchronization of neurons by caudally travelling waves of inhibition, the activity of interneurons was more coherent in the orthogonal mediolateral axis than in the rostrocaudal axis. If directed functional connectivity within the HVC is important for stereotyped, learned song, then it may be lacking in juveniles, which sing a highly variable song. Indeed, we found little evidence for network directionality in juveniles. These data indicate that a functionally directed network within the HVC matures during sensorimotor learning and may underlie vocal patterning.

[Long-term plasticity of HVC-RA synapses in adult male zebra finches].

Long-term synaptic plasticity is considered as a key part of the neural mechanism of learning and memory. The production of learned vocalization of male zebra finches is closely related to high vocal center (HVC)-robust nucleus of the arcopallium (RA) pathway. However, the long-term plasticity of HVC-RA synapses is unclear. This study investigated the long-term plasticity of HVC-RA synapses in adult male zebra finches through in vivo field potential recording. The results showed that physiologic stimulation, i.e., δ rhythmic stimulation and low frequency stimulation could not effectively induce long-term synaptic plasticity. The former leaded to no change of the amplitudes of evoked population spikes, and the latter induced short-term depression (STD) of the amplitudes of the second evoked population spikes caused by paired pulses. But high frequency stimulation induced long-term depression (LTD) of the amplitudes of evoked population spikes to show out long-term synaptic plasticity. These results suggest that LTD represents the long-term plasticity of HVC-RA synapses in adult male zebra finches, which may be a key part of the neural mechanism of vocal learning and memory and can explain the plasticity of adult song to some degree.

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.

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.

Persistent representation of juvenile experience in the adult songbird brain.

Juveniles sometimes learn behaviors that they cease to express as adults. Whether the adult brain retains a record of experiences associated with behaviors performed transiently during development remains unclear. We addressed this issue by studying neural representations of song in swamp sparrows, a species in which juveniles learn and practice many more songs than they retain in their adult vocal repertoire. We exposed juvenile swamp sparrows to a suite of tutor songs and confirmed that, although many tutor songs were imitated during development, not all copied songs were retained into adulthood. We then recorded extracellularly in the sensorimotor nucleus HVC in anesthetized sparrows to assess neuronal responsiveness to songs in the adult repertoire, tutor songs, and novel songs. Individual HVC neurons almost always responded to songs in the adult repertoire and commonly responded even more strongly to a tutor song. Effective tutor songs were not simply those that were acoustically similar to songs in the adult repertoire. Moreover, the strength of tutor song responses was unrelated to the number of times that the bird sang copies of those songs in juvenile or adult life. Notably, several neurons responded most strongly to a tutor song performed only rarely and transiently during juvenile life, or even to a tutor song for which we could find no evidence of ever having been copied. Thus, HVC neurons representing songs in the adult repertoire also appear to retain a lasting record of certain tutor songs, including those imitated only transiently.

Neuronal stability and drift across periods of sleep: premotor activity patterns in a vocal control nucleus of adult zebra finches.

How stable are neural activity patterns compared across periods of sleep? We evaluated this question in adult zebra finches, whose premotor neurons in the nucleus robustus arcopallialis (RA) exhibit sequences of bursts during daytime singing that are characterized by precise timing relative to song syllables. Each burst has a highly regulated pattern of spikes. We assessed these spike patterns in singing that occurred before and after periods of sleep. For about half of the neurons, one or more premotor bursts had changed after sleep, an average of 20% of all bursts across all RA neurons. After sleep, modified bursts were characterized by a discrete, albeit modest, loss of spikes with compensatory increases in spike intervals, but not changes in timing relative to the syllable. Changes in burst structure followed both interrupted bouts of sleep (1.5-3 h) and full nights of sleep, implicating sleep and not circadian cycle as mediating these effects. Changes in burst structure were also observed during the day, but far less frequently. In cases where multiple bursts in the sequence changed in a single cell, the sequence position of those bursts tended to cluster together. Bursts that did not show discrete changes in structure also showed changes in spike counts, but not biased toward losses. We hypothesize that changes in burst patterns during sleep represent active sculpting of the RA network, supporting auditory feedback-mediated song maintenance.