Tarciso Velho

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Song‐Induced Gene Expression: A Window on Song Auditory Processing and Perception

Abstract: We review here evidence that a large portion of the caudomedial telencephalon of songbirds, distinct from the song control circuit, is involved in the perceptual processing of birdsong. When songbirds hear song, a number of caudomedial pallial areas are activated, as revealed by expression of the activity‐dependent gene zenk. These areas, which include field L subfields L1 and L3, as well as the adjacent caudomedial nidopallium (NCM) and caudomedial mesopallium (CMM), are part of the central auditory pathway and constitute a lobule in the caudomedial aspect of the telencephalon. Several lines of evidence indicate that the neural circuits integrating this lobule are capable of performing the auditory processing of song based on fine acoustic features. Thus, this lobule is well positioned to mediate song perceptual processing and discrimination, which are required for vocal communication and vocal learning. Importantly, the zenk gene encodes a transcription factor linked to synaptic plasticity, and it regulates the expression of target genes associated with specific neuronal cell functions. The induction of zenk likely represents a key regulatory event in a gene cascade triggered by song and leading to neuronal plasticity. Thus, zenk may be linked to molecular and cellular mechanisms underlying experience‐dependent modification of song‐responsive circuits. In summary, songbirds possess an elaborate system for song perceptual processing and discrimination that potentially also subserves song‐induced neuronal plasticity and song memory formation. The continued use of a multidisciplinary approach that integrates molecular, anatomical, physiological and behavioral methodologies has the potential to provide further significant insights into the underlying neurobiology of the perceptual aspects of vocal communication and learning.

Co-induction of activity-dependent genes in songbirds

Song behavior in songbirds induces the expression of activity‐dependent genes in brain areas involved in perceptual processing, production and learning of song. This genomic response is thought to represent a link between neuronal activation and long‐term changes in song‐processing circuits of the songbird brain. Here we demonstrate that Arc, an activity‐regulated gene whose product has dendritic localization and is associated with synaptic plasticity, is rapidly induced by song in the brain of zebra finches. We show that, in the context of song auditory stimulation, Arc expression is induced in several telencephalic auditory areas, most prominently the caudomedial nidopallium and mesopallium, whereas in the context of singing, Arc is also induced in song control areas, namely nucleus HVC, used as a proper name, the robust nucleus of the arcopallium and the interface nucleus of the nidopallium. We also show that song‐induced Arc expression co‐localizes at the cellular level with those of the transcriptional regulators zenk and c‐fos, and that the song induction of these three genes is dependent on activation of the mitogen‐activated protein kinase signaling pathway. These findings provide evidence for an involvement of Arc in the brains response to birdsong. They also demonstrate that genes representing distinct genomic and cellular regulatory programs, namely early effectors and transcription factors, are co‐activated in the same neuronal cells by a naturally learned stimulus.

GABAergic neurons participate in the brain's response to birdsong auditory stimulation

Birdsong is a learned vocal behaviour that requires intact hearing for its development in juveniles and for its maintenance during adulthood. However, the functional organization of the brain circuits involved in the perceptual processing of song has remained obscure. Here we provide evidence that GABAergic mechanisms are an important component of these circuits and participate in the auditory processing of birdsong. We first cloned a zebra finch homologue of the gene encoding the 65‐kDa isoform of glutamic acid decarboxylase (zGAD‐65), a specific GABAergic marker, and conducted an expression analysis by in situ hybridization to identify GABAergic cells and to map their distribution throughout auditory telencephalic areas. The results showed that field L2, the caudomedial nidopallium (NCM) and the caudomedial mesopallium (CMM) contain a high number of GABAergic cells. Using patch‐clamp brain slice recordings, we found abundant GABAergic mIPSCs in NCM. Pharmacological antagonism of mIPSCs induced large EPSC bursts, suggesting that tonic inhibition helps to stabilize NCM against runaway excitation via activation of GABA‐A receptors. Next, using double fluorescence in situ hybridization and double immunocytochemical labelling, we demonstrated that large numbers of GABAergic cells in NCM and CMM show inducible expression of the transcriptional regulator ZENK in response to song auditory stimulation. These data provide direct evidence that GABAergic neurons in auditory brain regions are activated by song stimulation. Altogether, our results suggest that GABAergic mechanisms participate in auditory processing and perception, and might contribute to the memorization of birdsong.

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2–3 d.

Noradrenergic Control of Gene Expression and Long-Term Neuronal Adaptation Evoked by Learned Vocalizations in Songbirds

Norepinephrine (NE) is thought to play important roles in the consolidation and retrieval of long-term memories, but its role in the processing and memorization of complex acoustic signals used for vocal communication has yet to be determined. We have used a combination of gene expression analysis, electrophysiological recordings and pharmacological manipulations in zebra finches to examine the role of noradrenergic transmission in the brain’s response to birdsong, a learned vocal behavior that shares important features with human speech. We show that noradrenergic transmission is required for both the expression of activity-dependent genes and the long-term maintenance of stimulus-specific electrophysiological adaptation that are induced in central auditory neurons by stimulation with birdsong. Specifically, we show that the caudomedial nidopallium (NCM), an area directly involved in the auditory processing and memorization of birdsong, receives strong noradrenergic innervation. Song-responsive neurons in this area express α-adrenergic receptors and are in close proximity to noradrenergic terminals. We further show that local α-adrenergic antagonism interferes with song-induced gene expression, without affecting spontaneous or evoked electrophysiological activity, thus dissociating the molecular and electrophysiological responses to song. Moreover, α-adrenergic antagonism disrupts the maintenance but not the acquisition of the adapted physiological state. We suggest that the noradrenergic system regulates long-term changes in song-responsive neurons by modulating the gene expression response that is associated with the electrophysiological activation triggered by song. We also suggest that this mechanism may be an important contributor to long-term auditory memories of learned vocalizations.

Mesoscopic Patterns of Neural Activity Support Songbird Cortical Sequences

Time-locked sequences of neural activity can be found throughout the vertebrate forebrain in various species and behavioral contexts. From “time cells” in the hippocampus of rodents to cortical activity controlling movement, temporal sequence generation is integral to many forms of learned behavior. However, the mechanisms underlying sequence generation are not well known. Here, we describe a spatial and temporal organization of the songbird premotor cortical microcircuit that supports sparse sequences of neural activity. Multi-channel electrophysiology and calcium imaging reveal that neural activity in premotor cortex is correlated with a length scale of 100 µm. Within this length scale, basal-ganglia–projecting excitatory neurons, on average, fire at a specific phase of a local 30 Hz network rhythm. These results show that premotor cortical activity is inhomogeneous in time and space, and that a mesoscopic dynamical pattern underlies the generation of the neural sequences controlling song.

Unstable neurons underlie a stable learned behavior

Motor skills can be maintained for decades, but the biological basis of this memory persistence remains largely unknown. The zebra finch, for example, sings a highly stereotyped song that is stable for years, but it is not known whether the precise neural patterns underlying song are stable or shift from day to day. Here we demonstrate that the population of projection neurons coding for song in the premotor nucleus, HVC, change from day to day. The most dramatic shifts occur over intervals of sleep. In contrast to the transient participation of excitatory neurons, ensemble measurements dominated by inhibition persist unchanged even after damage to downstream motor nerves. These observations offer a principle of motor stability: spatiotemporal patterns of inhibition can maintain a stable scaffold for motor dynamics while the population of principal neurons that directly drive behavior shift from one day to the next.

Enriched expression and developmental regulation of the middle-weight neurofilament (NF-M) gene in song control nuclei of the zebra finch.

Songbirds evolved a complex set of dimorphic telencephalic nuclei that are essential for the learning and production of song. These nuclei, which together make up the oscine song control system, present several neurochemical properties that distinguish them from the rest of the telencephalon. Here we show that the expression of the gene encoding the middle‐weight neurofilament (NF‐M), an important component of the neuronal cytoskeleton and a useful tool for studying the cytarchitectonic organization of mammalian cortical areas, is highly enriched in large neurons within pallial song control nuclei (nucleus HVC, robustus nucleus of the arcopallium, and lateral magnocellular nucleus of the nidopallium) of male zebra finches (Taeniopygia guttata). We also show that this transcript is highly expressed in large neurons in the medulla, pons, midbrain, and thalamus. Moreover, we demonstrate that NF‐M expression in song control nuclei changes during postembryonic development, peaking during an early phase of the song‐learning period that coincides with the maturation of the song system. We did not observe changes in NF‐M expression in auditory areas or in song control nuclei in the contexts of hearing song or singing, although these contexts result in marked induction of the transcription factor ZENK. This observation suggests that NF‐M might not be under the regulatory control of ZENK in auditory areas or in song control nuclei. Overall, our data indicate that NF‐M is a neurochemical marker for pallial song control nuclei and provide suggestive evidence of an involvement of NF‐M in the development and/or maturation of the oscine song control system. J. Comp. Neurol. 500:477–497, 2007.

Cloning and expression analysis of retinoic acid receptors in the zebra finch brain.

The vitamin A derivative retinoic acid is produced postembryonically in discrete portions of the songbird brain, including some of the nuclei involved in song production and song learning, and its synthesis is required for the normal maturation of song behavior. To identify the brain targets for retinoic acid action, we cloned the zebra finch homologs of the α, β, and γ classes of retinoic acid receptors (RARs). In situ hybridization analysis revealed that the mRNAs for all three RARs are expressed at different levels in several brain areas, with a broader distribution than the mRNA for retinaldehyde‐specific aldehyde dehydrogenase (zRalDH), a retinoic acid‐synthesizing enzyme. Detectable RAR expression was found in all nuclei of the song control system, with the most marked expression occurring within the striatal song nucleus area X. These observations are consistent with a persistent action of retinoic acid in the postembryonic and adult songbird brain and provide further evidence for an involvement of retinoic acid signaling in the control of learned vocal behavior in a songbird species. They also suggest that the striatum is a major target of retinoic acid in songbirds. J. Comp. Neurol. 489:23–41, 2005.