Rudimentary substrates for vocal learning in a suboscine.

Vocal learning has evolved in only a few groups of mammals and birds. The key neuroanatomical and behavioural links bridging vocal learners and non-learners are still unknown. Here we show that a non-vocal-learning suboscine, the eastern phoebe, expresses neural and behavioural substrates that are associated with vocal learning in closely related oscine songbirds. In phoebes, a specialized forebrain region in the intermediate arcopallium seems homologous to the oscine song nucleus RA (robust nucleus of arcopallium) by its neural connections, expression of glutamate receptors and singing-dependent immediate-early gene expression. Lesion of this RA-like region induces subtle but consistent song changes. Moreover, the unlearned phoebe song unexpectedly develops through a protracted ontogeny. These features provide the first evidence of forebrain vocal-motor control in suboscines, which has not been encountered in other avian non-vocal-learners, and offer a potential configuration of brain and behaviour from which vocal learning might have evolved.

The zebra finch paradox: song is little changed, but number of neurons doubles.

New neurons are added to the high vocal center (HVC) of adult males in seasonally breeding songbirds such as the canary (Serinus canaria) that learns new songs in adulthood, and the song sparrow (Melospiza melodia) that does not. In both cases, the new neurons numerically replace others that have died, resulting in a seasonal fluctuation in HVC volume and neuron number. Peaks in neuronal replacement in both species occur in the fall when breeding is over and song is variable. New neurons are added, too, to the HVC of zebra finches (Taeniopygia guttata) that do not learn new songs in adulthood and whose song remains stereotyped throughout the year. Here, we show that, in contrast to the observations in seasonal songbirds, neurons added to the zebra finch HVC are not part of a replacement process. Rather, they lead to a doubling in the number of neurons that project from HVC to the robust nucleus of the arcopallium (RA). As this happens, HVC volume remains constant and the packing density of its neurons increases. The HVC-RA neurons are part of a descending pathway that carries the pattern of learned song; some HVC-RA neurons are also responsive to song playback. The addition of HVC-RA neurons happens in zebra finches housed singly, but becomes more acute if the birds are housed communally. We speculate that new neurons added to the adult HVC may help with the production or perception of learned song, or both.

The origins of vocal learning: New sounds, new circuits, new cells.

We do not know how vocal learning came to be, but it is such a salient trait in human evolution that many have tried to imagine it. In primates this is difficult because we are the only species known to possess this skill. Songbirds provide a richer and independent set of data. I use comparative data and ask broad questions: How does vocal learning emerge during ontogeny? In what contexts? What are its benefits? How did it evolve from unlearned vocal signals? How was brain anatomy altered to enable vocal learning? What is the relation of vocal learning to adult neurogenesis? No one has described yet a circuit or set of circuits that can master vocal learning, but this knowledge may soon be within reach. Moreover, as we uncover how birds encode their learned song, we may also come closer to understanding how we encode our thoughts.

Variable food begging calls are harbingers of vocal learning.

Vocal learning has evolved in only a few groups of mammals and birds. The developmental and evolutionary origins of vocal learning remain unclear. The imitation of a memorized sound is a clear example of vocal learning, but is that when vocal learning starts? Here we use an ontogenetic approach to examine how vocal learning emerges in a songbird, the chipping sparrow. The first vocalizations of songbirds, food begging calls, were thought to be innate, and vocal learning emerges later during subsong, a behavior reminiscent of infant babbling. Here we report that the food begging calls of male sparrows show several characteristics associated with learned song: male begging calls are highly variable between individuals and are altered by deafening; the production of food begging calls induces c-fos expression in a forebrain motor nucleus, RA, that is involved with the production of learned song. Electrolytic lesions of RA significantly reduce the variability of male calls. The male begging calls are subsequently incorporated into subsong, which in turn transitions into recognizable attempts at vocal imitation. Females do not sing and their begging calls are not affected by deafening or RA lesion. Our results suggest that, in chipping sparrows, intact hearing can influence the quality of male begging calls, auditory-sensitive vocal variability during food begging calls is the first step in a modification of vocal output that eventually culminates with vocal imitation.

The relationship between nature of social change, age, and position of new neurons and their survival in adult zebra finch brain.

Some kinds of neurons are spontaneously recruited in the intact, healthy adult brain, but the variables that affect their survival are not always clear. We show that in caudal nidopallium of adult male zebra finches, the rostrocaudal position of newly recruited neurons, their age (1 vs 3 months), and the nature of social change (complex vs simple) after the neurons were born affect their survival. Greater social complexity promoted the survival of younger new neurons, and the demise of older ones; a less marked social change promoted the survival of older new neurons. These effects were position dependent. We suggest that functional correlations between new neuron recruitment/survival and its inferred benefit to the animal might be better perceived when taking into account the position of cells, their age at the time of life style changes, and the nature and magnitude of the life style change.

A learning program that ensures prompt and versatile vocal imitation.

Here we show how a migratory songbird, the chipping sparrow (Spizella passerina), achieves prompt and precise vocal imitation. Juvenile chipping sparrow males develop five to seven potential precursor songs; the normal development of these songs requires intact hearing but not imitation from external models. The potential precursor songs conform with general species-typical song parameters but differ from the song of wild, adult territorial males. As chipping sparrow males return from migration to start their first breeding season, they settle close to an older adult. The young male then stops producing all but one of its precursor songs, retaining the one that most resembles that of its neighbor. This single song then becomes more variable and, in a matter of days, is altered to closely match the neighbor's song. This elegant solution ensures species specificity and promptness of imitation.

FnTm2, a novel brain-specific transcript, is dynamically expressed in the song learning circuit of the zebra finch.

Zebra finch males learn their song by imitation, a process influenced by social variables. The neural pathways for acquisition and production of learned song are known, but the cellular and molecular underpinnings are not. Here we describe a novel gene named "FnTm2" ("Phantom 2") that is predicted to encode a small protein (220 aa) with a single fibronectin type III domain and a single transmembrane domain. This gene shows great variability in its expression in song system neurons of the anterior forebrain pathway (AFP), a circuit that influences song discrimination and is necessary for normal song acquisition. AFP nuclei that express FnTm2 include the nucleus HVC (its Area X-projecting neurons), Area X, and LMAN (core and shell). FnTm2 expression does not correlate with singing behavior like the immediate early gene ZENK. It is expressed variably during sleeping hours and is not dependent on an intact song circuit. FnTm2's expression is sensitive to hearing, because in deafened birds its expression is substantially reduced in the core of LMAN. Furthermore, a comparison of FnTm2 expression between mice and zebra finches revealed a conserved pattern of expression in the "limbic system." We suggest that FnTm2 may be sensitive to affective and/or attentional states and thus may provide insights on how social variables influence the production and discrimination of learned vocalizations.

Genomic resources for songbird research and their use in characterizing gene expression during brain development.

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3' ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30-50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis.

Neuronal recruitment in adult zebra finch brain during a reproductive cycle.

Previous studies suggest that adult neurogenesis and neuronal replacement are related to the acquisition of new information. The present study supports this hypothesis by showing that there is an increase in new neuron recruitment in brains of adult male and female zebra finches that coincides with the need to memorize vocalizations of nestlings before they fledge. We counted [(3)H]-Thymidine labeled neurons 40 days after [(3)H]-Thymidine injections. These counts were made in the parents' brains at the time eggs hatched, at the time juveniles fledged and still needed parental care, and at the time juveniles were already independent. We focused on nidopallium caudale (NC), a brain region which plays a role in sound processing. Recruitment of new NC neurons increased at the time the young fledged, followed by a significant decrease when the young reached independence. We suggest that this increase enables parents to recognize their own young when they are still dependent on parental feeding, yet easily lost among other fledglings in the colony. We saw no such increase in neuronal recruitment in the olfactory bulb, suggesting anatomical specificity for the effect seen in NC. We also found a preliminary, positive correlation between number of fledglings and number of new NC neurons in the parents' brain at fledging, suggesting that the number of neurons recruited is sensitive to the number of young fledged.

High levels of new neuron addition persist when the sensitive period for song learning is experimentally prolonged.

Socially reared zebra finch males imitate a song they hear during posthatching days 30-65; during this time, many new neurons are added to the high vocal center (HVC), a forebrain nucleus necessary for the production of learned song. New neuron addition drops sharply after day 65, and no new songs are imitated. In contrast, male zebra finches reared in isolation from other males have more variable songs at day 65 and thereafter can still imitate new sounds (Eales, 1985). We show that, in isolate birds, a greater number of new neurons continues to be added to HVC during the next 85 d, and this number correlates with syllable variability. We suggest that new neuron addition and turnover facilitate song change and that this effect lingers when an expected learning event is delayed.

Expression profiling of intermingled long-range projection neurons harvested by laser capture microdissection.

Gene expression data are most useful if they can be associated with specific cell types. This is particularly so in an organ such as the brain, where many different cell types lie in close proximity to each other. We used zebra finches (Taeniopygia guttata), fluorescent tracers and laser capture microdissection (LCM) to collect projection neurons and their RNAs from two interspersed populations from the same animal. RNA amplified from each cell class was reverse transcribed, fluorescently labeled, and hybridized to cDNA microarrays of genes expressed in the zebra finch brain. We applied strict fold-expression criteria, supplemented by statistical analysis, to single out genes that showed the most extreme and consistent differential expression between the two cell classes. Confirmation of the true expression pattern of these genes was made by in situ hybridization and Taqman quantitative PCR (qPCR). High quality RNA was obtained, too, from backfilled neurons birth-dated with bromodeoxyuridine (BrdU). We also quantified changes in the levels of three genes after singing behavior using qPCR. Thus, we have brought together a combination of techniques allowing for the molecular profiling of intermingled populations of projection neurons of known connectivity, age and experience, which should constitute a powerful tool for CNS research.

Social and spatial changes induce multiple survival regimes for new neurons in two regions of the adult brain: An anatomical representation of time?

Male zebra finches reared in family groups were housed initially in small indoors cages with three other companions. At 4-5 months of age these birds were treated with [(3)H]-thymidine and then placed in large outdoors aviaries by themselves or with other zebra finches. Counts of new neurons were made 40, 60 and 150 days after the change in housing. Recruitment of new neurons in nidopallium caudale (NC) was higher than in the hippocampal complex (HC); but in both brain regions it was higher in communally housed birds than in birds housed singly, suggesting that the complexity of the social setting affects new neuron survival. In addition, the new neurons lived longer in rostral NC than in its caudal counterpart, and neuronal turnover was faster and more significant in NC than in HC. Albeit indirect, this may be the first suggestion that different parts of the brain upgrade memories at different time intervals, yielding an anatomical representation of time.

Variable rate of singing and variable song duration are associated with high immediate early gene expression in two anterior forebrain song nuclei.

The duration of songs and the intervals between these songs are more variable when wild, adult, free-ranging chipping sparrows sing at dawn than when they sing during the day. The more variable delivery is used to interact with males, and the stereotyped delivery is used to attract females. In captive birds, however, the variability observed at dawn persists during the day. We quantified the expression of an immediate early gene, ZENK, in wild and captive birds and found that the level of song-associated ZENK expression in two song nuclei, Area X and lMAN, was positively related to variability in song duration and intersong interval and could be dissociated from the social context in which the song occurred. Thus, a combination of field and laboratory approaches helped us identify nuclei, context, and behavioral features associated with a change in gene expression thought to be a marker of behavioral variability.

The neural basis of birdsong.

Songbirds represent an excellent model system for understanding the neural mechanisms underlying learning.

Replaceable neurons and neurodegenerative disease share depressed UCHL1 levels.

Might there be systematic differences in gene expression between neurons that undergo spontaneous replacement in the adult brain and those that do not? We first explored this possibility in the high vocal center (HVC) of male zebra finches by using a combination of neuronal tracers, laser capture microdissection, and RNA profiling. HVC has two kinds of projection neurons, one of which continues to be produced and replaced in adulthood. HVC neurons of the replaceable kind showed a consistent and robust underexpression of the deubiquitination gene ubiquitin carboxyl-terminal hydrolase (UCHL1) that is involved with protein degradation. Singing behavior, known to increase the survival of adult-born HVC neurons in birds, significantly up-regulated the levels of UCHL1 in the replaceable neurons but not in their equally active nonreplaceable counterparts. We then looked in the mouse brain and found relatively low UCHL1 expression in granule neurons of the hippocampus and olfactory bulb, two well characterized types of replaceable neurons in mammals. UCHL1 dysfunction has been associated with neurodegeneration in Parkinson's, Alzheimer's, and Huntington's disease patients. In all these instances, reduced UCHL1 function may jeopardize the survival of CNS neurons.

Freedom and rules: the acquisition and reprogramming of a bird's learned song.

Canary song is hierarchically structured: Short stereotyped syllables are repeated to form phrases, which in turn are arranged to form songs. This structure occurs even in the songs of young isolates, which suggests that innate rules govern canary song development. However, juveniles that had never heard normal song imitated abnormal synthetic songs with great accuracy, even when the tutor songs lacked phrasing. As the birds matured, imitated songs were reprogrammed to form typical canary phrasing. Thus, imitation and innate song constraints are separate processes that can be segregated in time: freedom in youth, rules in adulthood.

Juvenile zebra finches can use multiple strategies to learn the same song.

Does the ontogeny of vocal imitation follow a set program that, given a target sound, unfolds in a predictable manner, or is it more like problem solving, with many possible solutions? We report that juvenile male zebra finches, Taeniopygia guttata, can master their imitation of the same song in various ways; these developmental trajectories are sensitive to the social setting in which the bird grows up. A variety of vocal developmental trajectories have also been described in infants. Are these many ways to learn unique to the vocal domain or a hallmark of advanced brain function?

The road we travelled: discovery, choreography, and significance of brain replaceable neurons.

Neurons are constantly added to the telencephalon of songbirds. In the high vocal center (HVC), where this has been studied, new neurons replace older ones that died. Peaks in replacement are seasonal and affect some neuronal classes but not others. Peaks in replacement coincide with peaks in information acquisition. The new neurons are produced by division of cells in the wall of the lateral ventricle. Where studied closely, the neuronal stem cells proved to be radial glia. Life expectancy of the new neurons ranges from weeks to months. New neuron survival is regulated by vacancies, hormones, and activity. The immediate agent of new neuron survival is, in some cases, brain-derived neurotrophic factor (BDNF). The effect of BDNF is maximal 14-20 days after the cells are born, when they are establishing their connections. These observations are now being extended to other vertebrates and may apply, to varying degrees, to all of them. The function of neuronal replacement in healthy adult brain remains unclear. If synaptic number and efficacy sufficed as mechanisms for long-term memory storage and could be adjusted again and again to incorporate new memories, then neuronal replacement would seem unnecessary. Since it occurs, it seems reasonable to suppose that replacement serves to maintain learning potential in a way that could not be done just by synaptic change. Long-term memories may be encoded by long-term changes in gene expression akin to a last step in cell differentiation. If so, neuronal replacement may be the adult brain's way of striking a balance between limited memory space and the need to acquire new memories. The testing of this hypothesis remains in the future. This chapter tells how neuronal replacement was discovered in the adult songbird brain.

Age and experience affect the recruitment of new neurons to the song system of zebra finches during the sensitive period for song learning: ditto for vocal learning in humans?

Vocal learning in songbirds and humans is a complex learned skill with sensory, motor, and social aspects. It culminates in the imitation of sounds produced by other, usually older individuals. Song learning and language learning may differ in their cognitive content, but both require coordination of auditory feedback and fine motor control, which may be supported by similar brain structures. Vocal learning in birds as in humans requires the use of forebrain networks; in songbirds these networks are thought to be related, in part, to the frontal association cortex-basal ganglia loops that mature in humans at adolescence.