Models of vocal learning in the songbird: Historical frameworks and the stabilizing critic.

Birdsong is a form of sensorimotor learning that involves a mirror-like system that activates with both song hearing and production. Early models of song learning, based on behavioral measures, identified key features of vocal plasticity, such as the requirements for memorization of a tutor song and auditory feedback during song practice. The concept of a comparator, which compares the memory of the tutor song to auditory feedback, featured prominently. Later models focused on linking anatomically-defined neural modules to behavioral concepts, such as the comparator. Exploiting the anatomical modularity of the songbird brain, localized lesions illuminated mechanisms of the neural song system. More recent models have integrated neuronal mechanisms identified in other systems with observations in songbirds. While these models explain multiple aspects of song learning, they must incorporate computational elements based on unknown biological mechanisms to bridge the motor-to-sensory delay and/or transform motor signals into the sensory domain. Here, I introduce the stabilizing critic hypothesis, which enables sensorimotor learning by (1) placing a purely sensory comparator afferent of the song system and (2) endowing song system disinhibitory interneuron networks with the capacity both to bridge the motor-sensory delay through prolonged bursting and to stabilize song segments selectively based on the comparator signal. These proposed networks stabilize an otherwise variable signal generated by both putative mirror neurons and a cortical-basal ganglia-thalamic loop. This stabilized signal then temporally converges with a matched premotor signal in the efferent song motor cortex, promoting spike-timing-dependent plasticity in the premotor circuitry and behavioral song learning.

Rhythmic cortical neurons increase their oscillations and sculpt basal ganglia signaling during motor learning.

The function and modulation of neural circuits underlying motor skill may involve rhythmic oscillations (Feller, 1999; Marder and Goaillard, 2006; Churchland et al., 2012). In the proposed pattern generator for birdsong, the cortical nucleus HVC, the frequency and power of oscillatory bursting during singing increases with development (Crandall et al., 2007; Day et al., 2009). We examined the maturation of cellular activity patterns that underlie these changes. Single unit ensemble recording combined with antidromic identification (Day et al., 2011) was used to study network development in anesthetized zebra finches. Autocovariance quantified oscillations within single units. A subset of neurons oscillated in the theta/alpha/mu/beta range (8-20 Hz), with greater power in adults compared to juveniles. Across the network, the normalized oscillatory power in the 8-20 Hz range was greater in adults than juveniles. In addition, the correlated activity between rhythmic neuron pairs increased with development. We next examined the functional impact of the oscillators on the output neurons of HVC. We found that the firing of oscillatory neurons negatively correlated with the activity of cortico-basal ganglia neurons (HVC(X)s), which project to Area X (the song basal ganglia). If groups of oscillators work together to tonically inhibit and precisely control the spike timing of adult HVC(X)s with coordinated release from inhibition, then the activity of HVC(X)s in juveniles should be decreased relative to adults due to uncorrelated, tonic inhibition. Consistent with this hypothesis, HVC(X)s had lower activity in juveniles. These data reveal network changes that shape cortical-to-basal ganglia signaling during motor learning.

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.

Identification of single neurons in a forebrain network.

Behaviors are generated from complex interactions among networks of neurons. Single-unit ensemble recording has been used to identify multiple neurons in functioning networks. These recordings have provided insight into interactions among neurons in local and distributed circuits. Recorded units in these ensembles have been classed based on waveform type, firing pattern, and physical location. To identify individual projection neurons in a cortical network, we have paired tetrode recording with antidromic stimulation. We developed techniques that enable antidromic identification of single units and study of functional interactions between these neurons and other circuit elements. These methods have been developed in the zebra finch and should be applicable, with potential modifications that we discuss here, to any neural circuit with defined subpopulations based on projection target. This methodology will enable elucidation of the functional roles of single identified neurons in complex vertebrate circuits.

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.

Daily and developmental modulation of "premotor" activity in the birdsong system.

Human speech and birdsong are shaped during a sensorimotor sensitive period in which auditory feedback guides vocal learning. To study brain activity as song learning occurred, we recorded longitudinally from developing zebra finches during the sensorimotor phase. Learned sequences of vocalizations (motifs) were examined along with contemporaneous neural population activity in the song nucleus HVC, which is necessary for the production of learned song (Nottebohm et al. 1976: J Comp Neurol 165:457-486; Simpson and Vicario 1990: J Neurosci 10:1541-1556). During singing, HVC activity levels increased as the day progressed and decreased after a night of sleep in juveniles and adults. In contrast, the pattern of HVC activity changed on a daily basis only in juveniles: activity bursts became more pronounced during the day. The HVC of adults was significantly burstier than that of juveniles. HVC bursting was relevant to song behavior because the degree of burstiness inversely correlated with the variance of song features in juveniles. The song of juveniles degrades overnight (Deregnaucourt et al. 2005: Nature 433:710-716). Consistent with a relationship between HVC activity and song plasticity (Day et al. 2008: J Neurophys 100:2956-2965), HVC burstiness degraded overnight in young juveniles and the amount of overnight degradation declined with developmental song learning. Nocturnal changes in HVC activity strongly and inversely correlated with the next day's change, suggesting that sleep-dependent degradation of HVC activity may facilitate or enable subsequent diurnal changes. Collectively, these data show that HVC activity levels exhibit daily cycles in adults and juveniles, whereas HVC burstiness and song stereotypy change daily in juveniles only. In addition, the data indicate that HVC burstiness increases with development and inversely correlates with song variability, which is necessary for trial and error vocal learning.

Top-down regulation of plasticity in the birdsong system: "premotor" activity in the nucleus HVC predicts song variability better than it predicts song features.

We studied real-time changes in brain activity during active vocal learning in the zebra finch songbird. The song nucleus HVC is required for the production of learned song. To quantify the relationship of HVC activity and behavior, HVC population activity during repeated vocal sequences (motifs) was recorded and temporally aligned relative to the motif, millisecond by millisecond. Somewhat surprisingly, HVC activity did not reliably predict any vocal feature except amplitude and, to a lesser extent, entropy and pitch goodness (sound periodicity). Variance in "premotor" HVC activity did not reliably predict variance in behavior. In contrast, HVC activity inversely predicted the variance of amplitude, entropy, frequency, pitch, and FM. We reasoned that, if HVC was involved in song learning, the relationship of HVC activity to learned features would be developmentally regulated. To test this hypothesis, we compared the HVC song feature relationships in adults and juveniles in the sensorimotor "babbling" period. We found that the relationship of HVC activity to variance in FM was developmentally regulated, with the greatest difference at an HVC vocalization lag of 50 ms. Collectively, these data show that, millisecond by millisecond, bursts in HVC activity predict song stability on-line during singing, whereas decrements in HVC activity predict plasticity. These relationships between neural activity and plasticity may play a role in vocal learning in songbirds by enabling the selective stabilization of parts of the song that match a learned tutor model.

HVC neural sleep activity increases with development and parallels nightly changes in song behavior.

Sleep abnormalities are coexpressed with human communication disorders. Recent data from the birdsong system, the best model for human speech, indicate that sleep has a critical role in vocal learning. To understand the neural mechanisms that underlie behavioral changes during sleep, we recorded sleep activity in the song control area HVC longitudinally during song development in zebra finches. We focused on the sensorimotor phase of song learning, when the finch shapes his song behavior toward a learned tutor song model. Direct comparison of sleep activity in adults and juveniles revealed that the juvenile HVC has a lower spike rate and longer silent periods than the adult. Within individual finches, sleep silent periods decreased and spike rate increased with age. We next systematically compared neural sleep activity and song behavior. We now report that spike rate during sleep was significantly correlated with overnight changes in song behavior. Collectively, these data indicate that sleep activity in the vocal control area HVC increases with age and may affect song behavior.

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.

Neural auditory selectivity develops in parallel with song.

The zebra finch learns his song by memorizing a tutor's vocalization and then using auditory feedback to match his current vocalization to this memory, or template. The neural song system of adult and young birds responds to auditory stimuli, and exhibits selective tuning to the bird's own song (BOS). We have directly examined the development of neural tuning in the song motor system. We measured song system responses to vocalizations produced at various ages during sleep. We now report that the auditory response of the song motor system and motor output are linked early in song development. During sleep, playback of the current BOS induced a response in the song nucleus HVC during the song practice period, even when the song consisted of little more than repeated begging calls. Halfway through the sensorimotor period when the song was not yet in its final form, the response to BOS already exceeded that to all other auditory stimuli tested. Moreover, responses to previous, plastic versions of BOS decayed over time. This indicates that selective tuning to BOS mirrors the vocalization that the bird is currently producing.

Neural song preference during vocal learning in the zebra finch depends on age and state.

The zebra finch acquires its song by first memorizing a model song from a tutor and then matching its own vocalizations to the memory trace of the tutor song, called a template. Neural mechanisms underlying this process require a link between the neural memory trace and the premotor song circuitry, which drives singing. We now report that a premotor song nucleus responds more to the tutor song model than to every other stimulus examined, including the bird's own song (BOS). Neural tuning to the song model occurred only during waking and peaked during the template-matching period of development, when the vocal motor output is sculpted to match the tutor song. During the same developmental phase, the BOS was the most effective excitatory stimulus during sleep. The preference for BOS compared to tutor song inverted with sleep/wake state. Thus, song preference shifts with development and state.