Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

Synaptic and molecular mechanisms regulating plasticity during early learning.

Many behaviors are learned most easily during a discrete developmental period, and it is generally agreed that these "sensitive periods" for learning reflect the developmental regulation of molecular or synaptic properties that underlie experience-dependent changes in neural organization and function. Avian song learning provides one example of such temporally restricted learning, and several features of this behavior and its underlying neural circuitry make it a powerful model for studying how early experience sculpts neural and behavioral organization. Here we describe evidence that within the basal ganglia-thalamocortical loop implicated in vocal learning, song acquisition engages N-methyl-d-aspartate receptors (NMDARs), as well as signal transduction cascades strongly implicated in other instances of learning. Furthermore, NMDAR phenotype changes in parallel with developmental and seasonal periods for vocal plasticity. We also review recent studies in the avian song system that challenge the popular notion that sensitive periods for learning reflect developmental changes in the NMDAR that alter thresholds for synaptic plasticity.

LMAN lesions prevent song degradation after deafening without reducing HVC neuron addition.

In some songbirds perturbing auditory feedback can promote changes in song structure well beyond the end of song learning. One factor that may drive vocal change in such deafened birds is the ongoing addition of new vocal-motor neurons into the song system. Without auditory feedback to guide their incorporation, the addition of these new neurons could disrupt the established song pattern. To assess this hypothesis, the authors determined if neuronal recruitment into the vocal motor nucleus HVC is affected by neural signals that influence vocal change in adult deafened birds. Such signals appear to be conveyed via LMAN, a nucleus in the anterior forebrain that is necessary for vocal change after deafening. Here the authors tested whether LMAN lesions might restrict song degradation after deafening by reducing the addition or survival of new HVC neurons that would otherwise corrupt the ongoing song pattern. Using [3H]thymidine autoradiography to identify neurons generated in adult zebra finches, it was shown here that LMAN lesions do not reduce the number or percent of new HVC neurons surviving for either several weeks or months after [3H]thymidine labeling. However, the authors confirmed previous reports that LMAN lesions restrict vocal change after deafening. These data suggest that neurons incorporated into the adult HVC may form behaviorally adaptive connections without requiring auditory feedback, and that any role such neurons may play in promoting vocal change after adult deafening requires anterior forebrain pathway output.

Song tutoring triggers CaMKII phosphorylation within a specialized portion of the avian basal ganglia.

In several songbird species, a specialized anterior forebrain pathway (AFP) that includes part of the avian basal ganglia has been implicated specifically in song learning. To further elucidate cellular mechanisms and circuitry involved in vocal learning, we used quantitative immunoblot analysis to determine if early song tutoring promotes within the AFP phosphorylation of calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional kinase whose phosphorylation at threonine 286 is critical for many forms of neural plasticity and behavioral learning. We report that in young male zebra finches likely to have begun the process of song acquisition, brief tutoring by a familiar conspecific adult promotes a dramatic increase in levels of phosphorylated CaMKII (pCaMKII) in Area X, the striatal/pallidal component of the AFP. In contrast, pCaMKII levels in this region were not elevated if 1) the tutor did not sing, 2) the tutor sang but was visually isolated from the pupil, or 3) the tutor was an unfamiliar adult. In young males that had not previously heard any conspecific song, first exposure to a song tutor produced a more modest, but significant rise in pCaMKII levels. Young females (who do not develop song behavior) did not exhibit any effect of tutoring on pCaMKII levels in that portion of the basal ganglia that corresponds to Area X in males. These data are consistent with the hypothesis that Area X participates in encoding and/or attaching reward value to a representation of tutor song that is accessed later to guide motor learning.

Developmental patterns of NMDAR expression within the song system do not recur during adult vocal plasticity in zebra finches.

All songbirds learn to sing during postnatal development but then display species differences in the capacity to learn song in adulthood. While the mechanisms that regulate avian vocal plasticity are not well characterized, one contributing factor may be the composition of N-methyl-D-aspartate receptors (NMDAR). Previous studies of an anterior forebrain pathway implicated in vocal plasticity revealed significant regulation of NMDAR subunit expression during the developmental sensitive period for song learning. Much less is known about the developmental regulation of NMDAR subunit expression in regions that participate more directly in motor aspects of song behavior. We show here that an increase in NR2A subunit mRNA and a decrease in NR2B subunit mRNA within the vocal motor pathway accompany song learning in zebra finches; however, manipulations that can alter the timing of song learning did not alter the course of these developmental changes. We also tested whether adult deafening, a treatment that provokes vocal change in songbirds that normally sing a stable song throughout adulthood, would render NMDAR subunit expression more similar to that observed developmentally. We report that NR2A and NR2B mRNA levels did not change within the anterior forebrain or vocal motor pathways after adult deafening, even after substantial changes in song structure. These results indicate that vocal plasticity does not require "juvenile patterns" of NMDAR gene expression in the avian song system.