Learned Vocal Communication Research Articles

The role of cerebellum in learned vocal communication in adult songbirds

Injury, tumors, ischemia, and lesions in the cerebellum show the involvement of this region in human speech. The association of the cerebellum with learned birdsong has only been identified recently. Cerebellar dysfunction in young songbirds causes learning disabilities, but its role in adult songbirds has not been established. The aim of this study was to investigate the role of the deep cerebellar nuclei (DCN) in adult birdsong. We created bilateral excitotoxic lesions in the DCN of adult male zebra finches (Taeniopygia guttata) and recorded their songs for up to 4 months. Using magnetic resonance imaging (MRI) and immunohistochemistry, we validated the lesion efficacy. We found that the song duration significantly increased from 14 weeks post-op; the increase in duration was caused by a greater number of introductory notes as well as a greater number of syllables sung after the introductory notes. On the other hand, the motif duration decreased from 8 weeks after DCN lesions were induced, which was due to faster singing of syllables, not changes in inter-syllable interval length. DCN lesions also caused a decrease in the fundamental frequency of syllables. In summary, we showed that DCN lesions influence the temporal and acoustic features of birdsong. These results suggest that the cerebellum influences singing in adult songbirds.

Inhibition of miR-128 Enhances Vocal Sequence Organization in Juvenile Songbirds.

The molecular mechanisms underlying learned vocal communication are not well characterized. This is a major barrier for developing treatments for conditions affecting social communication, such as autism spectrum disorder (ASD). Our group previously generated an activity-dependent gene expression network in the striatopallidal song control nucleus, Area X, in adult zebra finches to identify master regulators of learned vocal behavior. This dataset revealed that the two host genes for microRNA-128, ARPP21 and R3HDM1, are among the top genes whose expression correlates to how much birds sing. Here we examined whether miR-128 itself is behaviorally regulated in Area X and found that its levels decline with singing. We hypothesized that reducing miR-128 during the critical period for vocal plasticity would enhance vocal learning. To test this, we bilaterally injected an antisense miR-128 construct (AS miR-128) or a control scrambled sequence into Area X at post-hatch day 30 (30 d) using sibling-matched experimental and control pupils. The juveniles were then returned to their home cage and raised with their tutors. Strikingly, inhibition of miR-128 in young birds enhanced the organization of learned vocal sequences. Tutor and pupil stereotypy scores were positively correlated, though the correlation was stronger between tutors and control pupils compared to tutors and AS miR-128 pupils. This difference was driven by AS miR-128 pupils achieving higher stereotypy scores despite their tutors’ lower syntax scores. AS miR-128 birds with tutors on the higher end of the stereotypy spectrum were more likely to produce songs with faster tempos relative to sibling controls. Our results suggest that low levels of miR-128 facilitate vocal sequence stereotypy. By analogy, reducing miR-128 could enhance the capacity to learn to speak in patients with non-verbal ASD. To our knowledge, this study is the first to directly link miR-128 to learned vocal communication and provides support for miR-128 as a potential therapeutic target for ASD.

Parallels in the sequential organization of birdsong and human speech

Human speech possesses a rich hierarchical structure that allows for meaning to be altered by words spaced far apart in time. Conversely, the sequential structure of nonhuman communication is thought to follow non-hierarchical Markovian dynamics operating over only short distances. Here, we show that human speech and birdsong share a similar sequential structure indicative of both hierarchical and Markovian organization. We analyze the sequential dynamics of song from multiple songbird species and speech from multiple languages by modeling the information content of signals as a function of the sequential distance between vocal elements. Across short sequence-distances, an exponential decay dominates the information in speech and birdsong, consistent with underlying Markovian processes. At longer sequence-distances, the decay in information follows a power law, consistent with underlying hierarchical processes. Thus, the sequential organization of acoustic elements in two learned vocal communication signals (speech and birdsong) shows functionally equivalent dynamics, governed by similar processes.

Auditory evoked BOLD responses in awake compared to lightly anaesthetized zebra finches

Functional magnetic resonance imaging (fMRI) is increasingly used in cognitive neuroscience and has become a valuable tool in the study of auditory processing in zebra finches, a well-established model of learned vocal communication. Due to its sensitivity to head motion, most fMRI studies in animals are performed in anaesthetized conditions, which might significantly impact neural activity evoked by stimuli and cognitive tasks. In this study, we (1) demonstrate the feasibility of fMRI in awake zebra finches and (2) explore how light anaesthesia regimes affect auditory-evoked BOLD responses to biologically relevant songs. After an acclimation procedure, we show that fMRI can be successfully performed during wakefulness, enabling the detection of reproducible BOLD responses to sound. Additionally, two light anaesthesia protocols were tested (isoflurane and a combination of medetomidine and isoflurane), of which isoflurane alone appeared to be the most promising given the high success rate, non-invasive induction, and quick recovery. By comparing auditory evoked BOLD responses in awake versus lightly anaesthetized conditions, we observed overall effects of anaesthetics on cerebrovascular reactivity as reflected in the extent of positive and negative BOLD responses. Further, our results indicate that light anaesthesia has limited effects on selective BOLD responses to natural versus synthetic sounds.

A reliable and flexible gene manipulation strategy in posthatch zebra finch brain

Songbird models meaningfully contribute to many fields including learned vocal communication, the neurobiology of social interactions, brain development, and ecology. The value of investigating gene-brain-behavior relationships in songbirds is therefore high. Viral infections typically used in other lab animals to deliver gene editing constructs have been less effective in songbirds, likely due to immune system properties. We therefore leveraged the in vivo electroporation strategy used in utero in rodents and in ovo in poultry, and apply it to posthatch zebra finch songbird chicks. We present a series of experiments with a combination of promoters, fluorescent protein genes, and piggyBac transposase vectors to demonstrate that this can be a reliable, efficient, and flexible strategy for genome manipulation. We discuss options for gene delivery experiments to test circuit and behavioral hypotheses using a variety of manipulations, including gene overexpression, CRISPR/Cas9 gene editing, inducible technologies, optogenetic or DREADD cellular control, and cell type-specific expression.

Molecular Microcircuitry Underlies Functional Specification in a Basal Ganglia Circuit Dedicated to Vocal Learning

Similarities between speech and birdsong make songbirds advantageous for investigating the neurogenetics of learned vocal communication--a complex phenotype probably supported by ensembles of interacting genes in cortico-basal ganglia pathways of both species. To date, only FoxP2 has been identified as critical to both speech and birdsong. We performed weighted gene coexpression network analysis on microarray data from singing zebra finches to discover gene ensembles regulated during vocal behavior. We found ∼2,000 singing-regulated genes comprising three coexpression groups unique to area X, the basal ganglia subregion dedicated to learned vocalizations. These contained known targets of human FOXP2 and potential avian targets. We validated biological pathways not previously implicated in vocalization. Higher-order gene coexpression patterns, rather than expression levels, molecularly distinguish area X from the ventral striato-pallidum during singing. The previously unknown structure of singing-driven networks enables prioritization of molecular interactors that probably bear on human motor disorders, especially those affecting speech.

Language-related Cntnap2 gene is differentially expressed in sexually dimorphic song nuclei essential for vocal learning in songbirds.

Multiple studies, involving distinct clinical populations, implicate contactin associated protein-like 2 (CNTNAP2) in aspects of language development and performance. While CNTNAP2 is broadly distributed in developing rodent brain, it shows a striking gradient of frontal cortical enrichment in developing human brain, consistent with a role in patterning circuits that subserve higher cognition and language. To test the hypothesis that CNTNAP2 may be important for learned vocal communication in additional species, we employed in situ hybridization to characterize transcript distribution in the zebra finch, an experimentally tractable songbird for which the neural substrate of this behavior is well established. Consistent with an important role in learned vocalization, Cntnap2 was enriched or diminished in key song control nuclei relative to adjacent brain tissue. Importantly, this punctuated expression was observed in males, but not females, in accord with the sexual dimorphism of neural circuitry and vocal learning in this species. Ongoing functional work will provide important insights into the relationship between Cntnap2 and vocal communication in songbirds and thereby clarify mechanisms at play in disorders of human cognition and language.

The Songbird as a Model for the Generation and Learning of Complex Sequential Behaviors

Over the past four decades songbirds have become a widely used model organism for neuroscientists studying complex sequential behaviors and sensory-guided motor learning. Like human babies, young songbirds learn many of the sounds they use for communication by imitating adults. This remarkable behavior emerges as a product of genetic predispositions and specific individual experiences. Research on different aspects of this behavior has elucidated key principles that may underlie vertebrate motor learning and motor performance in general, including (1) the mechanisms by which neural circuits generate sequential behaviors, (2) the existence of specialized neuronal circuits for the generation of exploratory variability, (3) the importance of basal ganglia-forebrain circuits for learning sequentially patterned behaviors, including speech and language, and (4) the existence of genetic toolkits that may have been coopted multiple times during evolution to play a role in learned vocal communication, such as the transcription factor FoxP2 and its molecular targets. This review presents new techniques, experiments, and findings in areas where songbirds have made significant contributions toward understanding of some of the most fundamental questions in neuroscience.

Neural correlates of categorical perception in learned vocal communication.

The division of continuously variable acoustic signals into discrete perceptual categories is a fundamental feature of vocal communication, including human speech. Despite the importance of categorical perception to learned vocal communication, the neural correlates underlying this phenomenon await identification. Here we report that individual sensorimotor neurons in freely behaving swamp sparrows express categorical auditory responses to changes in note duration, a learned feature of their songs, and that the neural response boundary accurately predicts the categorical perceptual boundary measured in field studies of the same sparrow population. Furthermore, swamp sparrow populations that learn different song dialects exhibit different categorical perceptual boundaries, consistent with the boundary being learned. Our results extend the analysis of the neural basis of perceptual categorization into the realm of vocal communication, while advancing the learned vocalizations of songbirds as a model for investigating how experience shapes categorical perception and the activity of categorically responsive neurons.

Inhibitory network interactions shape the auditory processing of natural communication signals in the songbird auditory forebrain.

The role of GABA in the central processing of complex auditory signals is not fully understood. We have studied the involvement of GABA A-mediated inhibition in the processing of birdsong, a learned vocal communication signal requiring intact hearing for its development and maintenance. We focused on caudomedial nidopallium (NCM), an area analogous to parts of the mammalian auditory cortex with selective responses to birdsong. We present evidence that GABA A-mediated inhibition plays a pronounced role in NCM's auditory processing of birdsong. Using immunocytochemistry, we show that approximately half of NCM's neurons are GABAergic. Whole cell patch-clamp recordings in a slice preparation demonstrate that, at rest, spontaneously active GABAergic synapses inhibit excitatory inputs onto NCM neurons via GABA A receptors. Multi-electrode electrophysiological recordings in awake birds show that local blockade of GABA A-mediated inhibition in NCM markedly affects the temporal pattern of song-evoked responses in NCM without modifications in frequency tuning. Surprisingly, this blockade increases the phasic and largely suppresses the tonic response component, reflecting dynamic relationships of inhibitory networks that could include disinhibition. Thus processing of learned natural communication sounds in songbirds, and possibly other vocal learners, may depend on complex interactions of inhibitory networks.

A Synaptic Basis for Auditory–Vocal Integration in the Songbird

Songbirds learn to sing by memorizing a tutor song that they then vocally mimic using auditory feedback. This developmental sequence suggests that brain areas that encode auditory memories communicate with brain areas for learned vocal control. In the songbird, the secondary auditory telencephalic region caudal mesopallium (CM) contains neurons that encode aspects of auditory experience. We investigated whether CM is an important source of auditory input to two sensorimotor structures implicated in singing, the telencephalic song nucleus interface (NIf) and HVC. We used reversible inactivation methods to show that activity in CM is necessary for much of the auditory-evoked activity that can be detected in NIf and HVC of anesthetized adult male zebra finches. Furthermore, extracellular and intracellular recordings along with spike-triggered averaging methods indicate that auditory selectivity for the bird's own song is enhanced between CM and NIf. We used lentiviral-mediated tracing methods to confirm that CM neurons directly innervate NIf. To our surprise, these tracing studies also revealed a direct projection from CM to HVC. We combined irreversible lesions of NIf with reversible inactivation of CM to establish that CM supplies a direct source of auditory drive to HVC. Finally, using chronic recording methods, we found that CM neurons are active in response to song playback and during singing, indicating their potential importance to song perception and processing of auditory feedback. These results establish the functional synaptic linkage between sites of auditory and vocal learning and may identify an important substrate for learned vocal communication.

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.

Genetic components of vocal learning.

Vocal learning is a rare trait. Humans depend on vocal learning to acquire spoken language, but most species that communicate acoustically have an innate repertoire of sounds that they use for information exchange. Among the few non-human species that also rely on vocal learning, songbirds have provided by far the most information for understanding this process. This article concentrates on the genetic components of vocal learning in humans and birds. We summarize the existing evidence for a genetic predisposition towards acquiring the species-specific human and avian vocal repertoires. We describe the approaches used for finding genes involved in shaping the neural circuitry required for vocal learning or in mediating the learning process itself. Special attention is given to a particular gene, FOXP2, which has been implicated in a human speech and language disorder. We have studied FoxP2 in avian vocal learners and non-learners and review evidence that links both the molecule and its close homologue FoxP1 to the development of brain regions implicated in vocal learning and to their function. FoxP2 has a characteristic expression pattern in a brain structure uniquely associated with learned vocal communication, Area X in songbirds, or its analogue in parrots and hummingbirds. In both avian song learners and non-learners FoxP2 expression predominates in sensory and sensory-motor circuits. These latter regions also express FoxP2 in mammals and reptiles. We conclude that FoxP2 is important for the building and function of brain pathways including, but not limited to, those essential for learned vocal communication.

Developmental regulation of basal ganglia circuitry during the sensitive period for vocal learning in songbirds.

A hallmark of sensitive periods of development is an enhanced capacity for learning, such that experience exerts a profound effect on the brain resulting in the establishment of behaviors and underlying neural circuitry that can last a lifetime. Songbirds, like humans, have a sensitive period for vocal learning: they acquire the sounds used for vocal communication during a restricted period of development. In principle, any organism that undertakes vocal learning is faced with the same challenge: to form some representation of target vocal sounds based on auditory experience, and then to translate that auditory target into a motor program that reproduces the sound. Both birds and humans achieve this translation by using auditory (and other) feedback resulting from incipient vocalizations ("babbling" in humans, "subsong" in birds) to adjust motor commands until vocal output produces a good copy of the target sounds. Similarities between vocal learning in birds and humans suggest that many aspects of the learning process have evolved to meet demands imposed by vocal communication. Thus songbirds provide a valuable animal model in which to study the physiological basis of learned vocal communication and the nature of sensitive periods in general. In this article, I describe aspects of both behavioral and neural frameworks that currently inform our thinking about mechanisms underlying vocal learning and behavior in songbirds, and highlight ideas that may need re-examination.

Selection-based learning in bird song development.

Bird song is a model system for study of the neurobiology, development, and functions of learned vocal communication signals. Research on avian song learning has been dominated by an instructive model of learning--the sensorimotor model. Developmental plasticity is assumed to be based on the incorporation of novel material into the song repertoire. Experimental evidence now indicates an alternative form of plasticity operating in harmony with sensorimotor learning, based on principles of selection, rather than instruction. Song dialects, a common consequence of vocal learning, can be achieved by overproduction of previously memorized songs and selective attrition of those that fail to match the dialect of interacting males. These distinct processes of learning have important implications for study of the neural substrates underlying song production and perception and for efforts to interpret patterns of geographic variation in song.