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.

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.

Peripheral androgen action helps modulate vocal production in a suboscine passerine.

Androgenic activation of intracellular androgen receptors (AR) influences avian vocal production, though this has largely been investigated at the level of the brain. We investigated the influence of predominantly peripheral AR on vocal output in wild Golden-collared Manakins (Manacus vitellinus). In this suboscine species, males court females by performing acrobatic displays and by producing relatively simple chee-poo vocalizations. To assess whether peripheral AR influences the acoustic structure of these vocal signals, we treated reproductively active adult males with the peripherally selective antiandrogen bicalutamide and then measured phonation performance. Inhibiting AR outside of the central nervous system increased the duration of the chee note and decreased the fundamental frequency of the poo note. This treatment caused no discernable change to chee-poo frequency modulation or entropy. Our results show that activation of peripheral AR mediates note-specific changes to temporal and pitch characteristics of the Golden-collared Manakin's main sexual call. Thus, our study provides one of the first demonstrations that androgenic action originating outside of the brain and likely on musculoskeletal targets can modulate avian vocal production.

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.