To transduce a zebra finch: interrogating behavioral mechanisms in a model system for speech.

The ability to alter neuronal gene expression, either to affect levels of endogenous molecules or to express exogenous ones, is a powerful tool for linking brain and behavior. Scientists continue to finesse genetic manipulation in mice. Yet mice do not exhibit every behavior of interest. For example, Mus musculus do not readily imitate sounds, a trait known as vocal learning and a feature of speech. In contrast, thousands of bird species exhibit this ability. The circuits and underlying molecular mechanisms appear similar between disparate avian orders and are shared with humans. An advantage of studying vocal learning birds is that the neurons dedicated to this trait are nested within the surrounding brain regions, providing anatomical targets for relating brain and behavior. In songbirds, these nuclei are known as the song control system. Molecular function can be interrogated in non-traditional model organisms by exploiting the ability of viruses to insert genetic material into neurons to drive expression of experimenter-defined genes. To date, the use of viruses in the song control system is limited. Here, we review prior successes and test additional viruses for their capacity to transduce basal ganglia song control neurons. These findings provide a roadmap for troubleshooting the use of viruses in animal champions of fascinating behaviors-nowhere better featured than at the 12th International Congress!

Behavior-linked FoxP2 regulation enables zebra finch vocal learning.

Mutations in the FOXP2 transcription factor cause an inherited speech and language disorder, but how FoxP2 contributes to learning of these vocal communication signals remains unclear. FoxP2 is enriched in corticostriatal circuits of both human and songbird brains. Experimental knockdown of this enrichment in song control neurons of the zebra finch basal ganglia impairs tutor song imitation, indicating that adequate FoxP2 levels are necessary for normal vocal learning. In unmanipulated birds, vocal practice acutely downregulates FoxP2, leading to increased vocal variability and dynamic regulation of FoxP2 target genes. To determine whether this behavioral regulation is important for song learning, here, we used viral-driven overexpression of FoxP2 to counteract its downregulation. This manipulation disrupted the acute effects of song practice on vocal variability and caused inaccurate song imitation. Together, these findings indicate that dynamic behavior-linked regulation of FoxP2, rather than absolute levels, is critical for vocal learning.

Expression analysis of the speech-related genes FoxP1 and FoxP2 and their relation to singing behavior in two songbird species.

Humans and songbirds are among the rare animal groups that exhibit socially learned vocalizations: speech and song, respectively. These vocal-learning capacities share a reliance on audition and cortico-basal ganglia circuitry, as well as neurogenetic mechanisms. Notably, the transcription factors Forkhead box proteins 1 and 2 (FoxP1, FoxP2) exhibit similar expression patterns in the cortex and basal ganglia of humans and the zebra finch species of songbird, among other brain regions. Mutations in either gene are associated with language disorders in humans. Experimental knock-down of FoxP2 in the basal ganglia song control region Area X during song development leads to imprecise copying of tutor songs. Moreover, FoxP2 levels decrease naturally within Area X when zebra finches sing. Here, we examined neural expression patterns of FoxP1 and FoxP2 mRNA in adult Bengalese finches, a songbird species whose songs exhibit greater sequence complexity and increased reliance on audition for maintaining their quality. We found that FoxP1 and FoxP2 expression in Bengalese finches is similar to that in zebra finches, including strong mRNA signals for both factors in multiple song control nuclei and enhancement of FoxP1 in these regions relative to surrounding brain tissue. As with zebra finches, when Bengalese finches sing, FoxP2 is behaviorally downregulated within basal ganglia Area X over a similar time course, and expression negatively correlates with the amount of singing. This study confirms that in multiple songbird species, FoxP1 expression highlights song control regions, and regulation of FoxP2 is associated with motor control of song.

Distinct neurogenomic states in basal ganglia subregions relate differently to singing behavior in songbirds.

Both avian and mammalian basal ganglia are involved in voluntary motor control. In birds, such movements include hopping, perching and flying. Two organizational features that distinguish the songbird basal ganglia are that striatal and pallidal neurons are intermingled, and that neurons dedicated to vocal-motor function are clustered together in a dense cell group known as area X that sits within the surrounding striato-pallidum. This specification allowed us to perform molecular profiling of two striato-pallidal subregions, comparing transcriptional patterns in tissue dedicated to vocal-motor function (area X) to those in tissue that contains similar cell types but supports non-vocal behaviors: the striato-pallidum ventral to area X (VSP), our focus here. Since any behavior is likely underpinned by the coordinated actions of many molecules, we constructed gene co-expression networks from microarray data to study large-scale transcriptional patterns in both subregions. Our goal was to investigate any relationship between VSP network structure and singing and identify gene co-expression groups, or modules, found in the VSP but not area X. We observed mild, but surprising, relationships between VSP modules and song spectral features, and found a group of four VSP modules that were highly specific to the region. These modules were unrelated to singing, but were composed of genes involved in many of the same biological processes as those we previously observed in area X-specific singing-related modules. The VSP-specific modules were also enriched for processes disrupted in Parkinson's and Huntington's Diseases. Our results suggest that the activation/inhibition of a single pathway is not sufficient to functionally specify area X versus the VSP and support the notion that molecular processes are not in and of themselves specialized for behavior. Instead, unique interactions between molecular pathways create functional specificity in particular brain regions during distinct behavioral states.

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.

Improved zebra finch brain transcriptome identifies novel proteins with sex differences.

The zebra finch (Taeniopygia guttata), a representative oscine songbird species, has been widely studied to investigate behavioral neuroscience, most notably the neurobiological basis of vocal learning, a rare trait shared in only a few animal groups including humans. In 2019, an updated zebra finch genome annotation (bTaeGut1_v1.p) was released from the Ensembl database and is substantially more comprehensive than the first version published in 2010. In this study, we utilized the publicly available RNA-seq data generated from Illumina-based short-reads and PacBio single-molecule real-time (SMRT) long-reads to assess the bird transcriptome. To analyze the high-throughput RNA-seq data, we adopted a hybrid bioinformatic approach combining short and long-read pipelines. From our analysis, we added 220 novel genes and 8,134 transcript variants to the Ensembl annotation, and predicted a new proteome based on the refined annotation. We further validated 18 different novel proteins by using mass-spectrometry data generated from zebra finch caudal telencephalon tissue. Our results provide additional resources for future studies of zebra finches utilizing this improved bird genome annotation and proteome.

Motor learning: the FoxP2 puzzle piece.

Mutation of the DNA-binding region of the FOXP2 protein causes an inherited language disorder. A recent study provides the first data on mice with this mutation, which exhibit deficits in motor-skill learning and abnormal properties of neural circuits that contribute to these skills.

Basal ganglia: Bursting with song.

The songs of mature zebra finches are notoriously repetitious, or 'crystallized'. Despite this stability, new work reveals that chronic pharmacologically driven bursting of cortical inputs to the basal ganglia can drive cumulative and lasting changes to multiple vocal features, including phenomena reminiscent of human stuttering.

Postnatal immune activation causes social deficits in a mouse model of tuberous sclerosis: Role of microglia and clinical implications.

There is growing evidence that prenatal immune activation contributes to neuropsychiatric disorders. Here, we show that early postnatal immune activation resulted in profound impairments in social behavior, including in social memory in adult male mice heterozygous for a gene responsible for tuberous sclerosis complex (Tsc2+/−), a genetic disorder with high prevalence of autism. Early postnatal immune activation did not affect either wild-type or female Tsc2+/− mice. We demonstrate that these memory deficits are caused by abnormal mammalian target of rapamycin­dependent interferon signaling and impairments in microglia function. By mining the medical records of more than 3 million children followed from birth, we show that the prevalence of hospitalizations due to infections in males (but not in females) is associated with future development of autism spectrum disorders (ASD). Together, our results suggest the importance of synergistic interactions between strong early postnatal immune activation and mutations associated with ASD.

Birdsong as a window into language origins and evolutionary neuroscience.

Humans and songbirds share the key trait of vocal learning, manifested in speech and song, respectively. Striking analogies between these behaviours include that both are acquired during developmental critical periods when the brain's ability for vocal learning peaks. Both behaviours show similarities in the overall architecture of their underlying brain areas, characterized by cortico-striato-thalamic loops and direct projections from cortical neurons onto brainstem motor neurons that control the vocal organs. These neural analogies extend to the molecular level, with certain song control regions sharing convergent transcriptional profiles with speech-related regions in the human brain. This evolutionary convergence offers an unprecedented opportunity to decipher the shared neurogenetic underpinnings of vocal learning. A key strength of the songbird model is that it allows for the delineation of activity-dependent transcriptional changes in the brain that are driven by learned vocal behaviour. To capitalize on this advantage, we used previously published datasets from our laboratory that correlate gene co-expression networks to features of learned vocalization within and after critical period closure to probe the functional relevance of genes implicated in language. We interrogate specific genes and cellular processes through converging lines of evidence: human-specific evolutionary changes, intelligence-related phenotypes and relevance to vocal learning gene co-expression in songbirds. This article is part of the theme issue 'What can animal communication teach us about human language?'

Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood.

In humans, mutations in the transcription factor forkhead box P2 (FOXP2) result in language disorders associated with altered striatal structure. Like speech, birdsong is learned through social interactions during maturational critical periods, and it relies on auditory feedback during initial learning and on-going maintenance. Hearing loss causes learned vocalizations to deteriorate in adult humans and songbirds. In the adult songbird brain, most FoxP2-enriched regions (e.g., cortex, thalamus) show a static expression level, but in the striatal song control nucleus, area X, FoxP2 is regulated by singing and social context: when juveniles and adults sing alone, its levels drop, and songs are more variable. When males sing to females, FoxP2 levels remain high, and songs are relatively stable: this "on-line" regulation implicates FoxP2 in ongoing vocal processes, but its role in the auditory-based maintenance of learned vocalization has not been examined. To test this, we overexpressed FoxP2 in both hearing and deafened adult zebra finches and assessed effects on song sung alone versus songs directed to females. In intact birds singing alone, no changes were detected between songs of males expressing FoxP2 or a GFP construct in area X, consistent with the marked stability of mature song in this species. In contrast, songs of males overexpressing FoxP2 became more variable and were less preferable to females, unlike responses to songs of GFP-expressing control males. In deafened birds, song deteriorated more rapidly following FoxP2 overexpression relative to GFP controls. Together, these experiments suggest that behavior-driven FoxP2 expression and auditory feedback interact to precisely maintain learned vocalizations.

Bidirectional scaling of vocal variability by an avian cortico-basal ganglia circuit.

Behavioral variability is thought to be critical for trial and error learning, but where such motor exploration is generated in the central nervous system is unclear. The zebra finch songbird species offers a highly appropriate model in which to address this question. The male song is amenable to detailed measurements of variability, while the brain contains an identified cortico-basal ganglia loop that underlies this behavior. We used pharmacogenetic interventions to separately interrogate cortical and basal ganglia nodes of zebra finch song control circuitry. We show that bidirectional manipulations of each node produce near mirror image changes in vocal control: Cortical activity promotes song variability, whereas basal ganglia activity promotes song stability. Furthermore, female conspecifics can detect these pharmacogenetically elicited changes in song quality. Our results indicate that cortex and striatopallidum can jointly and reciprocally affect behaviorally relevant levels of vocal variability, and point to endogenous mechanisms for its control.

FoxP2 isoforms delineate spatiotemporal transcriptional networks for vocal learning in the zebra finch.

Human speech is one of the few examples of vocal learning among mammals yet ~half of avian species exhibit this ability. Its neurogenetic basis is largely unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. We manipulated FoxP2 isoforms in Area X, a song-specific region of the avian striatopallidum analogous to human anterior striatum, during a critical period for song development. We delineate, for the first time, unique contributions of each isoform to vocal learning. Weighted gene coexpression network analysis of RNA-seq data revealed gene modules correlated to singing, learning, or vocal variability. Coexpression related to singing was found in juvenile and adult Area X whereas coexpression correlated to learning was unique to juveniles. The confluence of learning and singing coexpression in juvenile Area X may underscore molecular processes that drive vocal learning in young zebra finches and, by analogy, humans.

FoxP2 regulation during undirected singing in adult songbirds.

Learned vocal communication, including human speech, is a socially influenced behavior limited to certain animals. This ability requires auditory feedback during vocalization, which allows for on-line evaluation, to achieve the desired vocal output. To date, FOXP2 (forkhead box P2), a transcriptional repressor, is the only molecule directly linked to human speech. Identified FOXP2 mutations cause orofacial dyspraxia accompanied by abnormalities in corticostriatal circuitry controlling voluntary orofacial movements. These observations implicate FOXP2 in the developmental formation of neural circuits used in speech, but whether FOXP2 additionally plays an active role in mature circuitry was unknown. To address this question, we use a songbird, the zebra finch (Taeniopygia guttata), whose learned song and underlying circuitry are well characterized. We show that, when adult males sing, FoxP2 mRNA is acutely downregulated within area X, the specific region of the songbird striatum dedicated to song. Furthermore, we find downregulation in males that sing by themselves (undirected singers) but not in males that sing to females (directed singers). This FoxP2 downregulation cannot be a simple consequence of the motor act because birds sang in both directed and undirected contexts. Our data suggest that FoxP2 is important not only for the formation but also for the function of vocal control circuitry. Social context-dependent, acute changes in FoxP2 within the basal ganglia of adult songbirds also suggest, by analogy, that the core deficits of affected humans extend beyond development and beyond basic central motor control.

Singing mice, songbirds, and more: models for FOXP2 function and dysfunction in human speech and language.

In 2001, a point mutation in the forkhead box P2 (FOXP2) coding sequence was identified as the basis of an inherited speech and language disorder suffered by members of the family known as "KE." This mini-symposium review focuses on recent findings and research-in-progress, primarily from five laboratories. Each aims at capitalizing on the FOXP2 discovery to build a neurobiological bridge between molecule and phenotype. Below, we describe genetic through behavioral techniques used currently to investigate FoxP2 in birds, rodents, and humans for discovery of the neural bases of vocal learning and language.

Molecular evolution of PIII-SVMP and RGD disintegrin genes from the genus Crotalus.

Several types of disintegrins have been isolated from Crotalus spp rattlesnakes, including RGD disintegrins, and PIII-SVMPs. We isolated six cDNAs from snake venom glands using RT-PCR. Three RGD disintegrins (atroxatin, mojastin, and viridistatin) and three PIII-SVMPs (catroriarin, scutiarin, and viristiarin) cDNAs were isolated from the rattlesnakes Crotalus atrox, Crotalus scutulatus scutulatus, and Crotalus viridis viridis, respectively. Atroxatin and Viridistatin shared 90% amino acid identity to each other, and 87% identity to Mojastin. Scutiarin and Viristiarin were identical. All PIII-SVMPs isolated in this study shared the highest amino acid identity with Catrocollastatin. cDNA and protein sequences for RGD disintegrins, one MVD disintegrin, and PIII-SVMPs of the genus Crotalus (present in the NCBI database), were used in phylogenetic analysis. Neighbor-joining analysis of PIII-SVMP and RGD/MVD disintegrin-coding DNA sequences showed that these groups of genes separate into separate clades. A Phi(ST) pairwise comparison and Analysis of Molecular Variance (AMOVA) between PIII-SVMPs and RGD/MVD disintegrins showed significant genetic differences. Mutations observed in ten of the cDNAs analyzed did not affect Cys-coding sequences. Our K(A)/K(S) data suggest that rapid evolution occurred between the genes coding for PIII-SVMPs resulting, in the production of RGD disintegrin-coding genes. However, once these genes diverged, mutations in the PIII-SVMP-coding genes were accumulated less frequently.

A Sing-Song Way of Vocalizing: Generalization and Specificity in Language and Birdsong.

Spoken languages such as German are extremely discrete, whereas others such as Portuguese are melodic or "sing-song" wherein identifying a word relies on what comes before and after. Perhaps surprisingly, birdsong also exhibits specificity and generalization as articulated by Tian and Brainard (2017).

Parallel FoxP1 and FoxP2 expression in songbird and human brain predicts functional interaction.

Humans and songbirds are two of the rare animal groups that modify their innate vocalizations. The identification of FOXP2 as the monogenetic locus of a human speech disorder exhibited by members of the family referred to as KE enables the first examination of whether molecular mechanisms for vocal learning are shared between humans and songbirds. Here, in situ hybridization analyses for FoxP1 and FoxP2 in a songbird reveal a corticostriatal expression pattern congruent with the abnormalities in brain structures of affected KE family members. The overlap in FoxP1 and FoxP2 expression observed in the songbird suggests that combinatorial regulation by these molecules during neural development and within vocal control structures may occur. In support of this idea, we find that FOXP1 and FOXP2 expression patterns in human fetal brain are strikingly similar to those in the songbird, including localization to subcortical structures that function in sensorimotor integration and the control of skilled, coordinated movement. The specific colocalization of FoxP1 and FoxP2 found in several structures in the bird and human brain predicts that mutations in FOXP1 could also be related to speech disorders.