Neuronal Intrinsic Physiology Changes During Development of a Learned Behavior.

Juvenile male zebra finches learn their songs over distinct auditory and sensorimotor stages, the former requiring exposure to an adult tutor song pattern. The cortical premotor nucleus HVC (acronym is name) plays a necessary role in both learning stages, as well as the production of adult song. Consistent with neural network models where synaptic plasticity mediates developmental forms of learning, exposure to tutor song drives changes in the turnover, density, and morphology of HVC synapses during vocal development. A network's output, however, is also influenced by the intrinsic properties (e.g., ion channels) of the component neurons, which could change over development. Here, we use patch clamp recordings to show cell-type-specific changes in the intrinsic physiology of HVC projection neurons as a function of vocal development. Developmental changes in HVC neurons that project to the basal ganglia include an increased voltage sag response to hyperpolarizing currents and an increased rebound depolarization following hyperpolarization. Developmental changes in HVC neurons that project to vocal-motor cortex include a decreased resting membrane potential and an increased spike amplitude. HVC interneurons, however, show a relatively stable range of intrinsic features across vocal development. We used mathematical models to deduce possible changes in ionic currents that underlie the physiological changes and to show that the magnitude of the observed changes could alter HVC circuit function. The results demonstrate developmental plasticity in the intrinsic physiology of HVC projection neurons and suggest that intrinsic plasticity may have a role in the process of song learning.

Time-dependent localization of high- and low-sulfated keratan sulfates in the song nuclei of developing zebra finches.

Keratan sulfate proteoglycans (KSPGs) and chondroitin sulfate proteoglycans (CSPGs) consist of a protein core with covalently attached glycosaminoglycan side chain. Although CSPGs are known to regulate the end of the critical period, the role of KSPGs in brain development remains unclear. Young male zebra finches memorise song templates during development. The brain regions that are responsible for song learning, known as song nuclei, are recognized as a suitable model for the study of brain development. To understand the potential role of KSPGs, here we examined the localization of KSs with different degrees of sulfation in the brain of developing male zebra finches. Exclusively in the song nuclei, an increase in expression of 5-D-4-positive (5-D-4(+)) high-sulfated KS started after hatching, and reached a plateau at the end of the sensory period, during which the young bird listens to and memorises the song of an adult tutor. By contrast, weak and ubiquitous expression of BCD-4(+) low-sulfated KS remained unchanged until the end of the sensory period, and first increased in the song nuclei at the end of the sensorimotor period, during which the young bird produces plastic songs. Immunoblot analysis showed that phosphacan was a common core protein of 5-D-4(+) KS and BCD-4(+) KS. Finally, we confirmed that the sulfotransferase responsible for the synthesis of high-sulfated KS was exclusively localised in the song nuclei. Our observations suggest that time-dependent localization of KSPGs with different sulfation patterns in the song nuclei may underlie song learning in developing male zebra finches.

Identification of male-biased gene: parvalbumin in song control nuclei of the Bengalese finch.

Sex differences in song nuclei are evident across songbirds. To explore candidate genes involving in the sexual dimorphism of song nuclei, the present study used suppression subtraction hybridization to identify male-biased genes in the Bengalese finch (Lonchura striata). From 199 clones with an inserted sequence, we obtained a gene (parvalbumin, PV) coding a calcium-binding protein, which showed, through semi-quantitative PCR, obviously male-biased expression. In situ hybridization and immunohistochemistry indicated that PV was sexually distributed in most of the studied song nuclei, including in the high vocal center (HVC), the robust nucleus of the arcopallium (RA), Area X, and the lateral magnocellular nucleus of the anterior nidopallium (lMAN) for three studied age groups, namely, posthatching day (PD) 15, 45, and adult. The total number of PV mRNA or protein cells was significantly larger in males in the HVC, RA, and Area X for PD45 and adult. Considering that calcium-binding proteins have reported effects on the maturation of some brain areas, and on the sexual differentiation of mammalian brain areas by affecting cell survival rates, our study suggests that PV may be involved in the functional maturation of neurons in song nuclei or the sexual differentiation of song system.

Modulation of perineuronal nets and parvalbumin with developmental song learning.

Neural circuits and behavior are shaped during developmental phases of maximal plasticity known as sensitive or critical periods. Neural correlates of sensory critical periods have been identified, but their roles remain unclear. Factors that define critical periods in sensorimotor circuits and behavior are not known. Birdsong learning in the zebra finch occurs during a sensitive period similar to that for human speech. We now show that perineuronal nets, which correlate with sensory critical periods, surround parvalbumin-positive neurons in brain areas that are dedicated to singing. The percentage of both total and parvalbumin-positive neurons with perineuronal nets increased with development. In HVC (this acronym is the proper name), a song area important for sensorimotor integration, the percentage of parvalbumin neurons with perineuronal nets correlated with song maturity. Shifting the vocal critical period with tutor song deprivation decreased the percentage of neurons that were parvalbumin positive and the relative staining intensity of both parvalbumin and a component of perineuronal nets. Developmental song learning shares key characteristics with sensory critical periods, suggesting shared underlying mechanisms.

Conservation and expression of IQ-domain-containing calpacitin gene products (neuromodulin/GAP-43, neurogranin/RC3) in the adult and developing oscine song control system.

Songbirds are appreciated for the insights they provide into regulated neural plasticity. Here, we describe the comparative analysis and brain expression of two gene sequences encoding probable regulators of synaptic plasticity in songbirds: neuromodulin (GAP-43) and neurogranin (RC3). Both are members of the calpacitin family and share a distinctive conserved core domain that mediates interactions between calcium, calmodulin, and protein kinase C signaling pathways. Comparative sequence analysis is consistent with known phylogenetic relationships, with songbirds most closely related to chicken and progressively more distant from mammals and fish. The C-terminus of neurogranin is different in birds and mammals, and antibodies to the protein reveal high expression in adult zebra finches in cerebellar Purkinje cells, which has not been observed in other species. RNAs for both proteins are generally abundant in the telencephalon yet markedly reduced in certain nuclei of the song control system in adult canaries and zebra finches: neuromodulin RNA is very low in RA and HVC (relative to the surrounding pallial areas), whereas neurogranin RNA is conspicuously low in Area X (relative to surrounding striatum). In both cases, this selective downregulation develops in the zebra finch during the juvenile song learning period, 25-45 days after hatching. These results suggest molecular parallels to the robust stability of the adult avian song control circuit.

Top-down regulation of plasticity in the birdsong system: "premotor" activity in the nucleus HVC predicts song variability better than it predicts song features.

We studied real-time changes in brain activity during active vocal learning in the zebra finch songbird. The song nucleus HVC is required for the production of learned song. To quantify the relationship of HVC activity and behavior, HVC population activity during repeated vocal sequences (motifs) was recorded and temporally aligned relative to the motif, millisecond by millisecond. Somewhat surprisingly, HVC activity did not reliably predict any vocal feature except amplitude and, to a lesser extent, entropy and pitch goodness (sound periodicity). Variance in "premotor" HVC activity did not reliably predict variance in behavior. In contrast, HVC activity inversely predicted the variance of amplitude, entropy, frequency, pitch, and FM. We reasoned that, if HVC was involved in song learning, the relationship of HVC activity to learned features would be developmentally regulated. To test this hypothesis, we compared the HVC song feature relationships in adults and juveniles in the sensorimotor "babbling" period. We found that the relationship of HVC activity to variance in FM was developmentally regulated, with the greatest difference at an HVC vocalization lag of 50 ms. Collectively, these data show that, millisecond by millisecond, bursts in HVC activity predict song stability on-line during singing, whereas decrements in HVC activity predict plasticity. These relationships between neural activity and plasticity may play a role in vocal learning in songbirds by enabling the selective stabilization of parts of the song that match a learned tutor model.

Dietary retinoic acid affects song maturation and gene expression in the song system of the zebra finch.

Vitamin A, an essential nutrient, is required in its acidic form (retinoic acid) for normal embryogenesis and neuronal development, typically within well-defined concentration ranges. In zebra finches, a songbird species, localized retinoic acid synthesis in the brain is important for the development of song, a learned behavior sharing significant commonalities with speech acquisition in humans. We tested how dietary retinoic acid affects the development of song behavior and the brain's system for song control. Supplemental doses of retinoic acid given to juveniles during the critical period for song learning resulted in more variable or plastic-like songs when the birds reached adulthood, compared to the normal songs of vehicle-fed controls. We also observed that several genes (brinp1, nrgn, rxr-alpha, and sdr2/scdr9) had altered levels of expression in specific nuclei of the song system when comparing the experimental and control diet groups. Interestingly, we found significant correlations between gene expression levels in nuclei of the anterior forebrain pathway (lMAN and area X) and the degree of variability in the recorded songs. We observed, however, no major morphological effects such as changes in the volumes of song nuclei. Overall, our results lend further support to a fundamental role of retinoic acid in song maturation and point to possible molecular pathways associated with this action. The data also demonstrate that dietary content of Vitamin A can affect the maturation of a naturally learned complex behavior.

Hemispheric coordination is necessary for song production in adult birds: implications for a dual role for forebrain nuclei in vocal motor control.

Precise coordination across hemispheres is a critical feature of many complex motor circuits. In the avian song system the robust nucleus of the arcopallium (RA) plays a key role in such coordination. It is simultaneously the major output structure for the descending vocal motor pathway, and it also sends inputs to structures in the brain stem and thalamus that project bilaterally back to the forebrain. Because all birds lack a corpus callosum and the anterior commissure does not interconnect any of the song control nuclei directly, these bottom-up connections form the only pathway that can coordinate activity across hemispheres. In this study, we show that unilateral lesions of RA in adult male zebra finches (Taeniopigia guttata) completely and permanently disrupt the bird's stereotyped song. In contrast, lesions of RA in juvenile birds do not prevent the acquisition of normal song as adults. These results highlight the importance of hemispheric interdependence once the circuit is established but show that one hemisphere is sufficient for complex vocal behavior if this interdependence is prevented during a critical period of development. The ability of birds to sing with a single RA provides the opportunity to test the effect of targeted microlesions in RA without confound of functional compensation from the contralateral RA. We show that microlesions cause significant changes in song temporal structure and implicate RA as playing a major part in the generation of song temporal patterns. These findings implicate a dual role for RA, first as part of the program generator for song and second as part of the circuit that mediates interhemispheric coordination.

Plasticity of the Rufous-winged Sparrow, Aimophila carpalis, song control regions during the monsoon-associated summer breeding period.

In most temperate zone songbirds, exposure to increasing photoperiod in the spring stimulates the reproductive system and induces reproductive behaviors. Additionally, the brain regions that control singing (song control regions; SCRs) are larger during the breeding season, thus paralleling changes in the activity of the reproductive system. However, in some birds, environmental factors other than photoperiod initiate breeding. For example, free-living male Rufous-winged Sparrows develop their testes in March due to increasing photoperiod, but have relatively low plasma T until after they begin to breed, usually in July, during the monsoon period when day length is declining. We tested the hypothesis that SCRs grow and singing behavior increases after the monsoon rains begin. We captured adult male Rufous-winged Sparrows in July 2002, 7 days before and 20 days after the monsoon rains began, euthanized birds in the field, collected their brains, and measured SCR volumes from sections immunostained for the neuronal marker NeuN. In June and July 2006, we measured song rates in the field before and after the monsoon rains. SCR volumes were larger and singing behavior increased after the onset of the monsoon rains, coinciding with the initiation of breeding. Unlike in other species studied so far, SCR volumes grew as day length was decreasing. Comparative studies utilizing species that do not breed when day length is increasing may provide information on the relative contributions of various environmental factors to SCR neuroplasticity.

Increasing stereotypy in adult zebra finch song correlates with a declining rate of adult neurogenesis.

Adult neurogenesis is often correlated with learning new tasks, suggesting that a function of incorporating new neurons is to permit new memory formation. However, in the zebra finch, neurons are added to the song motor pathway throughout life, long after the initial song motor pattern is acquired by about 3 months of age. To explore this paradox, we examined the relationship between adult song structure and neuron addition using sensitive measures of song acoustic structure. We report that between 4 and 15 months of age there was an increase in the stereotypy of fine-grained spectral and temporal features of syllable acoustic structure. These results indicate that the zebra finch continues to refine motor output, perhaps by practice, over a protracted period beyond the time when song is first learned. Over the same age range, there was a decrease in the addition of new neurons to HVC, a region necessary for song production, but not to Area X or the hippocampus, regions not essential for singing. We propose that age-related changes in the stereotypy of syllable acoustic structure and HVC neuron addition are functionally related.

Song and brain development in canaries raised under different conditions of acoustic and social isolation over two years.

Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large normal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a significant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no significant change in repertoire size. Nor did they show any significant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year.

Developmental origin and identity of song system neurons born during vocal learning in songbirds.

New neurons are added to the forebrain song control regions high vocal center (HVC) and Area X of juvenile songbirds but the identity and site of origin of these cells have not been fully characterized. We used oncoretroviral vectors to genetically label neuronal progenitors in different regions of the zebra finch lateral ventricle. A region corresponding to the mammalian medial and lateral ganglionic eminences generated medium spiny neurons found in Area X and in the striatum surrounding Area X, and at least two classes of interneurons found in HVC. In addition, our experiments indicate that the HVC projection neurons that project into nucleus robust nucleus of the arcopallium (RA) are born locally from the ventricular region immediately dorsal to HVC. The ability to genetically target neuron subpopulations that give rise to different song system cell types provides a tool for specific genetic manipulations of these cell types. In addition, our results suggest striking similarities between neurogenesis in the embryonic mammalian brain and in the brain of the juvenile songbird and provide further evidence for the existence of conserved cell types in the forebrain for birds and mammals.

Activities of 3beta-HSD and aromatase in slices of developing and adult zebra finch brain.

Sex steroids influence the development and function of the songbird brain. Developmentally, the neural circuitry underlying song undergoes masculine differentiation under the influence of estradiol. In adults, estradiol stimulates song behavior and the seasonal growth of song control circuits. There is good reason to believe that these neuroactive estrogens are synthesized in the brain. At all ages, estrogens could act at the lateral ventricle, during migration, or where song nuclei exist or will form. We investigated the activity of two critical steroidogenic enzymes, 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and aromatase, using a slice culture system. Sagittal brain slices were collected from juvenile (posthatch day 20) and adult zebra finches containing either the lateral ventricle, where neurons are born, or the telencephalic song nuclei HVC and RA. The slices were incubated with (3)H-dehydroepiandrosterone or (3)H-androstenedione. Activity was determined by isolating certain products of 3beta-HSD (5alpha-androstanedione, 5beta-androstanedione, estrone, and estradiol) and aromatase (estrone and estradiol). Activities of both 3beta-HSD and aromatase were detected in all slices and were confirmed using specific enzyme inhibitors. We found no significant difference in activity between adult males and females in either region for either enzyme. Juvenile female slices containing the lateral ventricle, however, showed greater levels of 3beta-HSD activity than did similar slices from age-matched males. Determination of the activity of these critical steroidogenic enzymes in slice culture has implications for the role of neurosteroids in brain development.

Developmental modulation of the temporal relationship between brain and behavior.

Humans and songbirds shape learned vocalizations during a sensorimotor sensitive period or "babbling" phase. The brain mechanisms that underlie the shaping of vocalizations by sensory feedback are not known. We examined song behavior and brain activity in zebra finches during singing as they actively shaped their song toward a tutor model. We now show that the temporal relationship of behavior and activity in the premotor area HVC changes with the development of song behavior. During sensorimotor learning, HVC bursting activity both preceded and followed learned vocalizations by hundreds of milliseconds. Correspondingly, the duration of bursts that occurred during ongoing song motif behavior was prolonged in juveniles, as compared with adults, and was inversely correlated with song maturation. Multielectrode single-unit recording in juveniles revealed that single fast-spiking neurons were active both before and after vocalization. These same neurons responded to auditory stimuli. Collectively, these data indicate that a key aspect of sensory critical periods--prolonged bursting--also applies to sensorimotor development. In addition, prolonged motor discharge and sensory input coincide in single neurons of the developing song system, providing the necessary cellular elements for sensorimotor shaping through activity-dependent mechanisms.

Singing-related activity of identified HVC neurons in the zebra finch.

High vocal center (HVC) is part of the premotor pathway necessary for song production and is also a primary source of input to the anterior forebrain pathway (AFP), a basal ganglia-related circuit essential for vocal learning. We have examined the activity of identified HVC neurons of zebra finches during singing. Antidromic activation was used to identify three classes of HVC cells: neurons projecting to the premotor nucleus RA, neurons projecting to area X in the AFP, and putative HVC interneurons. HVC interneurons are active throughout the song and display tonic patterns of activity. Projection neurons exhibit highly phasic stereotyped firing patterns. X-projecting (HVC((X))) neurons burst zero to four times per motif, whereas RA-projecting neurons burst extremely sparsely--at most once per motif. The bursts of HVC projection neurons are tightly locked to the song and typically have a jitter of <1 ms. Population activity of interneurons, but not projection neurons, was significantly correlated with syllable patterns. Consistent with the idea that HVC codes for the temporal order in the song rather than for sound, the vocal dynamics and neural dynamics in HVC occur on different and uncorrelated time scales. We test whether HVC((X)) neurons are auditory sensitive during singing. We recorded the activity of these neurons in juvenile birds during singing and found that firing patterns of these neurons are not altered by distorted auditory feedback, which is known to disrupt learning or to cause degradation of song already learned.

Afferent influences on cell death and birth during development of a cortical nucleus necessary for learned vocal behavior in zebra finches.

Forebrain nuclei that control learned vocal behavior in zebra finches are anatomically distinct and interconnected by a simple pattern of axonal pathways. In the present study, we examined afferent regulation of neuronal survival during development of the robust nucleus of the archistriatum (RA). RA projection neurons form the descending motor pathway of cortical vocal-control regions and are believed to be directly involved in vocal production.RA receives afferent inputs from two other cortical regions, the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and the higher vocal center (HVC).However, because the ingrowth of HVC afferent input is delayed, lMAN projection neurons provide the majority of afferent input to RA during early vocal learning. lMAN afferent input to RA is of particular interest because lMAN is necessary for vocal learning only during a restricted period of development. By making lesions of lMAN in male zebra finches at various stages of vocal development (20-60 days of age) and in adults (>90-days old), we asked whether the survival of RA neurons depends on lMAN afferent input, and if so whether such dependence changes over the course of vocal learning. The results showed that removal of lMAN afferent input induced the loss of over 40% of RA neurons among birds in early stages of vocal development(20 days of age). However, lMAN lesions lost the ability to induce RA neuron death among birds in later stages of vocal development (40 days of age and older). These findings indicate that many RA neurons require lMAN afferent input for their survival during early vocal learning, whereas the inability of lMAN lesions to induce RA neuron death in older birds may indicate a reduced requirement for afferent input or perhaps the delayed ingrowth of HVC afferent input (at approx. 35 days of age)provides an alternate source of afferent support. Removal of lMAN afferent input also dramatically increased the incidence of mitotic figures in RA, but only among 20-day-old birds at 2 days post-lesion. The early, acute nature of the mitotic events raises the possibility that cell division in RA may be regulated by lMAN afferent input.