A reafferent and feed-forward model of song syntax generation in the Bengalese finch

Adult Bengalese finches generate a variable song that obeys a distinct and individual syntax. The syntax is gradually lost over a period of days after deafening and is recovered when hearing is restored. We present a spiking neuronal network model of the song syntax generation and its loss, based on the assumption that the syntax is stored in reafferent connections from the auditory to the motor control area. Propagating synfire activity in the HVC codes for individual syllables of the song and priming signals from the auditory network reduce the competition between syllables to allow only those transitions that are permitted by the syntax. Both imprinting of song syntax within HVC and the interaction of the reafferent signal with an efference copy of the motor command are sufficient to explain the gradual loss of syntax in the absence of auditory feedback. The model also reproduces for the first time experimental findings on the influence of altered auditory feedback on the song syntax generation, and predicts song- and species-specific low frequency components in the LFP. This study illustrates how sequential compositionality following a defined syntax can be realized in networks of spiking neurons

Long-Distance Retinoid Signaling in the Zebra Finch Brain

All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, is a powerful signaling molecule that regulates large-scale morphogenetic processes during vertebrate embryonic development, but is also involved post-natally in regulating neural plasticity and cognition. In songbirds, it plays an important role in the maturation of learned song. The distribution of the ATRA-synthesizing enzyme, zRalDH, and of ATRA receptors (RARs) have been described, but information on the distribution of other components of the retinoid signaling pathway is still lacking. To address this gap, we have determined the expression patterns of two obligatory RAR co-receptors, the retinoid X receptors (RXR) α and γ, and of the three ATRA-degrading cytochromes CYP26A1, CYP26B1, and CYP26C1. We have also studied the distribution of zRalDH protein using immunohistochemistry, and generated a refined map of ATRA localization, using a modified reporter cell assay to examine entire brain sections. Our results show that (1) ATRA is more broadly distributed in the brain than previously predicted by the spatially restricted distribution of zRalDH transcripts. This could be due to long-range transport of zRalDH enzyme between different nuclei of the song system: Experimental lesions of putative zRalDH peptide source regions diminish ATRA-induced transcription in target regions. (2) Four telencephalic song nuclei express different and specific subsets of retinoid-related receptors and could be targets of retinoid regulation; in the case of the lateral magnocellular nucleus of the anterior nidopallium (lMAN), receptor expression is dynamically regulated in a circadian and age-dependent manner. (3) High-order auditory areas exhibit a complex distribution of transcripts representing ATRA synthesizing and degrading enzymes and could also be a target of retinoid signaling. Together, our survey across multiple connected song nuclei and auditory brain regions underscores the prominent role of retinoid signaling in modulating the circuitry that underlies the acquisition and production of learned vocalizations

Network mechanisms underlying stable motor actions

While we can learn to produce stereotyped movements and maintain this ability for years, it is unclear how populations of individual neurons change their firing properties to coordinate these skills. This has been difficult to address because there is a lack of tools that can monitor populations of single neurons in freely behaving animals for the durations required to remark on their tuning. This thesis is divided into two main directions- device engineering and systems neuroscience. The first section describes the development of an electrode array comprised of tiny self-splaying carbon fibers that are small and flexible enough to avoid the immune response that typically limits electrophysiological recordings. I also describe the refinement of a head-mounted miniature microscope system, optimized for multi-month monitoring of cells expressing genetically encoded calcium indicators in freely behaving animals. In the second section, these tools are used to answer basic systems neuroscience questions in an animal with one of the most stable, complex learned behaviors in the animal kingdom: songbirds. This section explores the functional organization and long-term network stability of HVC, the songbird premotor cortical microcircuit that controls song. Our results reveal that neural activity in HVC is correlated with a length scale of 100um. At this mesocopic scale, basal-ganglia projecting excitatory neurons, on average, fire at a specific phase of a local 30Hz 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. At this mesoscopic level, neural coding is stable for weeks and months. These ensemble patterns persist after peripheral nerve damage, revealing that sensory-motor correspondence is not required to maintain the stability of the underlying neural ensemble. However, closer examination of individual excitatory neurons reveals that the participation of cells can change over the timescale of days- with particularly large shifts occurring over instances of sleep. Our findings suggest that fine-scale drift of projection neurons, stabilized by mesoscopic level dynamics dominated by inhibition, forms the mechanistic basis of memory maintenance and and motor stability

Network dynamics in the neural control of birdsong

Sequences of stereotyped actions are central to the everyday lives of humans and animals, from the kingfisher's dive to the performance of a piano concerto. Lashley asked how neural circuits managed this feat nearly 6 decades ago, and to this day it remains a fundamental question in neuroscience. Toward answering this question, vocal performance in the songbird was used as a model to study the performance of learned, stereotyped motor sequences. The first component of this work considers the song motor cortical zone HVC in the zebra finch, an area that sends precise timing signals to both the descending motor pathway, responsible for stereotyped vocal performance in the adult, and the basal ganglia, which is responsible for both motor variability and song learning. Despite intense interest in HVC, previous research has exclusively focused on describing the activity of small numbers of neurons recorded serially as the bird sings. To better understand HVC network dynamics, both single units and local field potentials were sampled across multiple electrodes simultaneously in awake behaving zebra finches. The local field potential and spiking data reveal a stereotyped spatio-temporal pattern of inhibition operating on a 30 ms time-scale that coordinates the neural sequences in principal cells underlying song. The second component addresses the resilience of the song circuit through cutting the motor cortical zone HVC in half along one axis. Despite this large-scale perturbation, the finch quickly recovers and sings a near-perfect song within a single day. These first two studies suggest that HVC is functionally organized to robustly generate neural dynamics that enable vocal performance. The final component concerns a statistical study of the complex, flexible songs of the domesticated canary. This study revealed that canary song is characterized by specific long-range correlations up to 7 seconds long-a time-scale more typical of human music than animal vocalizations. Thus, the neural sequences underlying birdsong must be capable of generating more structure and complexity than previously thought

Molecular profiling of sex-specific development of song and the song control nucleus HVC of songbirds

Singing of songbird species is a behavior that integrates multiple sensory inputs and motor outputs, which primarily rely on interconnected neural circuits in the avian brain, the song control system. The nucleus HVC is of particular interest because of its integrative roles in the song control system. In the majority of Northern temperate songbird species, males sing predominately, and they often use their songs to attract female mates. In contrast, most Northern temperate female songbirds sing either rarely or only in certain contexts. The characteristics and functions of female songs are less clear. In the tropics, many species of females sing regularly and depending on species, their song complexity is less or comparable to that of males. However, the HVC volume is greater in males than females in all songbird species that have been examined. In the first experiment of this thesis (p. 43), I described song features of spontaneously and rarely singing female canaries (Serinus canaria), a Northern temperate songbird species. I observed higher blood testosterone concentrations and greater HVC volume of singing females than that of non-singing females. The results suggest female canary singing is testosterone-dependent. Subcutaneous testosterone implantation induces singing in female canaries. In the second experiment (p. 65), I implanted female canaries with testosterone for six time periods (T1h, T3h, T8h, T3d, T7d, and T14d) and studied changes of gene expression in the HVC. I observed approximately 2,600 genes regulated by testosterone after one hour and the regulation was dynamic throughout the experimental time window. I investigated putative biological functions of testosterone-regulated genes in the six time points by gene ontology (GO)-term enrichment analysis, and showed that the enrichment of angiogenesis began at T1h and the enrichment of neurogenesis began at T3h, with both processes continuing until T14d. Furthermore, genes associated with “GABA” and “spine” were enriched in T3d birds when the birds started singing, while the number of genes associated with “nervous system development” was highest in T14d birds, when the HVC volume was significantly greater than controls. Finally, using approaches integrating gene expression, HVC volume, circulating testosterone levels, and song characteristics, I identified a potential master regulator of testosterone-regulated changes. Male canary songs vary seasonally. Breeding season songs are longer, louder, and more complex than non-breeding season songs. Non-breeding males implanted with testosterone sing songs resembling that of breeding season songs. In the third experiment (p. 81), I studied gene expression in the HVC of seven canary groups, females and males of breeding season and of non-breeding season, non-breeding season females and males treated with testosterone, and spontaneously singing female canaries. Hierarchical clustering and principal component analysis (PCA) showed that circulating testosterone levels and sex were the predominant variables associated with variation in the HVC transcriptomes. Comparison between natural singing canaries with testosterone-induced singing canaries of the same sex revealed large differences in the HVC transcriptomes. Moreover, the intersection of natural and testosterone-induced singing females shared little resemblance with males in terms of genes. GO-term enrichment analysis suggested functional overlap between sex-specific gene networks. However, although strong transcriptional changes in HVC correlate with the transition from non-singing to singing in both sexes, the type of transcriptional changes are sex-specific. In the fourth experiment (p. 89), I studied sex differences in HVC gene expression between three songbird species: the canary, the blue-capped cordon bleus (Uraeginthus cyanocephalus), and the forest weavers (Ploceus bicolor). Cordon bleu females sing regularly with female-specific songs, whereas forest weaver females sing songs identical to males. I found substantial sex differences in HVC gene expression in all three species, and sex-biased genes differed between species. Surprisingly, the majority of sex-biased genes were on autosomes instead the sex chromosome Z. These results provide further evidence for sex differences in brain structure at the molecular and cellular levels in sexually reproductive animals

Linear and Nonlinear Auditory Response Properties Of Interneurons In A High Order Avian Vocal Motor Nucleus During Wakefulness

Motor-related forebrain areas in higher vertebrates also show responses to passively presented sensory stimuli. Sensory tuning properties in these areas, especially during wakefulness, and their relation to perception, however, are poorly understood. In the avian song system, HVC (proper name) is a vocal-motor structure with auditory responses well defined under anesthesia but poorly characterized during wakefulness. I used a large set of song stimuli including the bird‟s own song (BOS) and many conspecific stimuli (CON) to characterize auditory tuning properties in putative interneurons (HVCIN) during wakefulness. My findings suggest that HVC contains a heterogeneity of response types; a third of neurons are either suppressed or show no response to any stimuli and two thirds show excitatory responses to one or more stimuli. A subset of excitatory neurons are tuned exclusively to BOS and show very low linearity as measured by spectrotemporal receptive field analysis (STRF), but many respond well to both BOS and CON stimuli and show response linearity comparable to that previously measured in structures of the ascending auditory pathway. Fourier analysis of the STRFs of linear HVCIN reveals a range of peak spectrotemporal tuning properties, with approximately half of these neurons showing peak sensitivity to modulations occurring with high power in zebra finch song. Previous work has established that HVC lesioned birds are impaired in operant contingency reversals involving CON stimuli and birds with lesions to song nuclei receiving auditory input from HVC are impaired in discriminations between BOS and CON stimuli. The findings of the present study are consistent with these results and suggest a possible role for HVC in species-relevant auditory tasks

Molecular profiling of sex-specific development of song and the song control nucleus HVC of songbirds

Singing of songbird species is a behavior that integrates multiple sensory inputs and motor outputs, which primarily rely on interconnected neural circuits in the avian brain, the song control system. The nucleus HVC is of particular interest because of its integrative roles in the song control system. In the majority of Northern temperate songbird species, males sing predominately, and they often use their songs to attract female mates. In contrast, most Northern temperate female songbirds sing either rarely or only in certain contexts. The characteristics and functions of female songs are less clear. In the tropics, many species of females sing regularly and depending on species, their song complexity is less or comparable to that of males. However, the HVC volume is greater in males than females in all songbird species that have been examined. In the first experiment of this thesis (p. 43), I described song features of spontaneously and rarely singing female canaries (Serinus canaria), a Northern temperate songbird species. I observed higher blood testosterone concentrations and greater HVC volume of singing females than that of non-singing females. The results suggest female canary singing is testosterone-dependent. Subcutaneous testosterone implantation induces singing in female canaries. In the second experiment (p. 65), I implanted female canaries with testosterone for six time periods (T1h, T3h, T8h, T3d, T7d, and T14d) and studied changes of gene expression in the HVC. I observed approximately 2,600 genes regulated by testosterone after one hour and the regulation was dynamic throughout the experimental time window. I investigated putative biological functions of testosterone-regulated genes in the six time points by gene ontology (GO)-term enrichment analysis, and showed that the enrichment of angiogenesis began at T1h and the enrichment of neurogenesis began at T3h, with both processes continuing until T14d. Furthermore, genes associated with “GABA” and “spine” were enriched in T3d birds when the birds started singing, while the number of genes associated with “nervous system development” was highest in T14d birds, when the HVC volume was significantly greater than controls. Finally, using approaches integrating gene expression, HVC volume, circulating testosterone levels, and song characteristics, I identified a potential master regulator of testosterone-regulated changes. Male canary songs vary seasonally. Breeding season songs are longer, louder, and more complex than non-breeding season songs. Non-breeding males implanted with testosterone sing songs resembling that of breeding season songs. In the third experiment (p. 81), I studied gene expression in the HVC of seven canary groups, females and males of breeding season and of non-breeding season, non-breeding season females and males treated with testosterone, and spontaneously singing female canaries. Hierarchical clustering and principal component analysis (PCA) showed that circulating testosterone levels and sex were the predominant variables associated with variation in the HVC transcriptomes. Comparison between natural singing canaries with testosterone-induced singing canaries of the same sex revealed large differences in the HVC transcriptomes. Moreover, the intersection of natural and testosterone-induced singing females shared little resemblance with males in terms of genes. GO-term enrichment analysis suggested functional overlap between sex-specific gene networks. However, although strong transcriptional changes in HVC correlate with the transition from non-singing to singing in both sexes, the type of transcriptional changes are sex-specific. In the fourth experiment (p. 89), I studied sex differences in HVC gene expression between three songbird species: the canary, the blue-capped cordon bleus (Uraeginthus cyanocephalus), and the forest weavers (Ploceus bicolor). Cordon bleu females sing regularly with female-specific songs, whereas forest weaver females sing songs identical to males. I found substantial sex differences in HVC gene expression in all three species, and sex-biased genes differed between species. Surprisingly, the majority of sex-biased genes were on autosomes instead the sex chromosome Z. These results provide further evidence for sex differences in brain structure at the molecular and cellular levels in sexually reproductive animals