Transgenic songbirds offer an opportunity to develop a genetic model for vocal learning.

Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus encoded the GFP regulated by the human ubiquitin-C promoter [Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Science 295:868-872], which is active in a wide variety of cells. The virus was injected into the very early embryo (blastodisc stage) to target the primordial germline cells that later give rise to sperm and eggs. A total of 265 fertile eggs were injected with virus, and 35 hatched (13%); 23 of these potential founders (F0) were bred, and three (13%) produced germline transgenic hatchlings that expressed the GFP protein (F1). Two of these three founders (F0) have produced transgenic young at a rate of 12% and the third at a rate of 6%. Furthermore, two of the F1 generation transgenics have since reproduced, one having five offspring (all GFP positive) and the other four offsping (one GFP positive).

A brain for all seasons: cyclical anatomical changes in song control nuclei of the canary brain.

Male canaries that have reached sexual maturity can, in subsequent years, learn new song repertoires. Two telencephalic song control nuclei, the hyperstriatum ventrale, pars caudale, and nucleus robustus archistriatalis are, respectively, 99 and 76 percent larger in the spring, when male canaries are producing stable adult song, than in the fall, at the end of the molt and after several months of not singing. It is hypothesized that such fluctuations reflect an increase and then reduction in numbers of synapses and are related to the yearly ability to acquire new motor coordinations.

Auditory responses in avian vocal motor neurons: a motor theory for song perception in birds.

The hypoglossal motor neurons that innervate the vocal organ (syrinx) of the male zebra finch show a selective, long-latency (50-millisecond) response to sound. This response is eliminated by lesions to forebrain song-control nuclei. Different song syllables elicit a response from different syringeal motor neurons. Conspecific vocalizations may therefore be perceived as members of a set of vocal gestures and thus distinct from other environmental sounds. This hypothesis is an avian parallel to the motor theory of speech perception in humans.

Gonadal hormones induce dendritic growth in the adult avian brain.

Ovariectomized adult female canaries were treated with physiological doses of testosterone, dihydrotestosterone, or estradiol. Singing, which is typical of males, occurred in the testosterone-treated birds but not in any of the other birds. The effect of these hormones was assessed on dendrites from a class of neurons in the nucleus robustus archistriatalis (RA), a forebrain nucleus for song control. The RA neurons of the testosterone-treated birds had dendritic trees resembling those of intact males. The RA neurons of the estradiol- and dihydrotesterone-treated birds resembled those of intact females. All hormone-treated groups had dendrites that were significantly longer than those of untreated ovariectomized females. Thus gonadal hormones induce dendritic growth in the adult avian brain.

Sexual dimorphism in vocal control areas of the songbird brain.

In canaries and zebra finches, three vocal control areas in the brain are strikingly larger in males than in females. A fourth, area X of the lobus parolfactorius, is well developed in males of both species, less well developed in femal canaries, and absent or not recognizable in femal zebra finches. These size differences correlate well with differences in singing behavior. Males of both species learn song by reference to auditory information, and females do not normally sing. Exogenous testosterone induces singing in female canaries but not in female zebra finches. This is believed to be the first report of such gross sexual dimorphism in a vertebrate brain.

Quantal duration of auditory memories.

Neuronal responses in the caudomedial neostriatum (NCM) of adult zebra finches (Taeniopygia guttata) decreased upon repeated, unreinforced presentations of conspecific song, calls, or other complex sounds. This "stimulus-specific habituation" is a form of learning, and its spontaneous loss, a form of "forgetting." Spontaneous forgetting occurred only at narrowly defined times (2 to 3, 6 to 7, 14 to 15, 17 to 18.5, 46 to 48, or 85 to 89 hours after first exposure to a stimulus), determined by stimulus class, number of presentations, and interval between presentations. The first five forgetting times coincided with periods when gene expression and protein synthesis in NCM were required for maintenance of the longer lasting (85 to 89 hours) habituation. The number of successive episodes of gene expression induced by a stimulus, but occurring long after stimulus presentation, appears to determine the quantal duration of auditory memories.

Birth of projection neurons in adult avian brain may be related to perceptual or motor learning.

Projection neurons that form part of the motor pathway for song control continue to be produced and to replace older projection neurons in adult canaries and zebra finches. This is shown by combining [3H]thymidine, a cell birth marker, and fluorogold, a retrogradely transported tracer of neuronal connectivity. Species and seasonal comparisons suggest that this process is related to the acquisition of perceptual or motor memories. The ability of an adult brain to produce and replace projection neurons should influence our thinking on brain repair.

Androgen-concentrating cells in the midbrain of a songbird.

Androgen-concentrating cells were found in the midbrain of the chaffinch Fringilla coelebs by autoradiography using tritiated testosterone. Labeled cells were localized primarily in the nucleus intercollicularis, an area from which vocalizations can be electrically stimulated in birds. These autoradiographic results suggest that the nucleus intercollicularis is a site in the action of androgens on avian vocal behavior.

Neurons generated in the adult brain are recruited into functional circuits.

Adult canaries, Serinus canarius, received injections of 3H-labeled thymidine, a marker of DNA synthesis. Thirty days after the last injection, intracellular potentials were recorded from neurons in the nucleus hyperstriatum ventralis pars caudalis, a vocal control nucleus in the telencephalon; these same cells were then injected with horseradish peroxidase. Of the 74 neurons labeled with horseradish peroxidase that were recovered, the nuclei of seven were radioactively labeled. Four of these seven neurons had responded to auditory stimuli. These double-labeled neurons were apparently generated during or after the 3H-labeled thymidine treatment (during adulthood) and subsequently incorporated into functional neural circuits.

Dynamics of the vocal imitation process: how a zebra finch learns its song.

Song imitation in birds provides good material for studying the basic biology of vocal learning. Techniques were developed for inducing the rapid onset of song imitation in young zebra finches and for tracking trajectories of vocal change over a 7-week period until a match to a model song was achieved. Exposure to a model song induced the prompt generation of repeated structured sounds (prototypes) followed by a slow transition from repetitive to serial delivery of syllables. Tracking this transition revealed two phenomena: (i) Imitations of dissimilar sounds can emerge from successive renditions of the same prototype, and (ii) developmental trajectories for some sounds followed paths of increasing acoustic mismatch until an abrupt correction occurred by period doubling. These dynamics are likely to reflect underlying neural and articulatory constraints on the production and imitation of sounds.

BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain.

New neurons are incorporated into the high vocal center (HVC), a nucleus of the adult canary (Serinus canaria) brain that plays a critical role in the acquisition and production of learned song. Recruitment of new neurons in the HVC is seasonally regulated and depends upon testosterone levels. We show here that brain-derived neurotrophic factor (BDNF) is present in the HVC of adult males but is not detectable in that of females, though the HVC of both sexes has BDNF receptors (TrkB). Testosterone treatment increases the levels of BDNF protein in the female HVC, and BDNF infused into the HVC of adult females triples the number of new neurons. Infusion of a neutralizing antibody to BDNF blocks the testosterone-induced increase in new neurons. Our results demonstrate that BDNF is involved in the regulation of neuronal replacement in the adult canary brain and suggest that the effects of testosterone are mediated through BDNF.

Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division.

Neurogenesis in the adult avian brain is restricted to the telencephalon. New neurons originate in the ventricular zone (VZ) from cells that have not been identified. We mapped the position of [3H]thymidine-labeled cells in the walls of the ventricles of the adult canary brain. Labeled VZ cells were restricted to the telencephalon (lateral ventricles) and concentrated in "hot spots". The coincidence of these hot spots with regions rich in radial cells suggested that radial cells may be the cells undergoing mitosis. We used smears prepared from fragments of the VZ containing the hot spots to show directly that radial cells accumulate [3H]thymidine. In addition, grain counts at different survival times demonstrated that these cells divide. Hot spots of VZ cell division also coincided with sites of neuronal origin. We suggest that radial cell division may give rise to new neurons.

For whom the bird sings: context-dependent gene expression.

Male zebra finches display two song behaviors: directed and undirected singing. The two differ little in the vocalizations produced but greatly in how song is delivered. "Directed" song is usually accompanied by a courtship dance and is addressed almost exclusively to females. "Undirected" song is not accompanied by the dance and is produced when the male is in the presence of other males, alone, or outside a nest occupied by its mate. Here, we show that the anterior forebrain vocal pathway contains medial and lateral "cortical-basal ganglia" subdivisions that have differential ZENK gene activation depending on whether the bird sings female-directed or undirected song. Differences also occur in the vocal output nucleus, RA. Thus, although these two vocal behaviors are very similar, their brain activation patterns are dramatically different.

Site-specific retinoic acid production in the brain of adult songbirds.

The song system of songbirds, a set of brain nuclei necessary for song learning and production, has distinctive morphological and functional properties. Utilizing differential display, we searched for molecular components involved in song system regulation. We identified a cDNA (zRalDH) that encodes a class 1 aldehyde dehydrogenase. zRalDH was highly expressed in various song nuclei and synthesized retinoic acid efficiently. Brain areas expressing zRalDH generated retinoic acid. Within song nucleus HVC, only projection neurons not undergoing adult neurogenesis expressed zRalDH. Blocking zRalDH activity in the HVC of juveniles interfered with normal song development. Our results provide conclusive evidence for localized retinoic acid synthesis in an adult vertebrate brain and indicate that the retinoic acid-generating system plays a significant role in the maturation of a learned behavior.

Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds.

In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.

Probes for rare mRNAs reveal distributed cell subsets in canary brain.

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.

Reassessing the mechanisms and origins of vocal learning in birds.

The most widely accepted hypothesis of vocal imitation in birds pre-dates many recent studies on the behavior, anatomy, physiology and cell biology of this phenomenon. It states that vocal learning involves two steps: (1) an auditory memory is laid down, and then (2) vocal output is modified until the auditory feedback it generates matches the model. This black-box model of vocal imitation disregards circuitry. We now know that the brain pathways for vocal learning in birds include a series of well-defined nuclei and projections. Some of these nuclei and projections develop late in ontogeny, at the time when auditory models are first acquired and imitated. We also know that the pathways involved in song production respond to sound, an observation that blurs the demarcation between what is an auditory and what is a motor circuit. These and other recent discoveries call for a reassessment of the mechanisms and origins of vocal learning in birds and mammals.

Age at deafening affects the stability of learned song in adult male zebra finches.

Male zebra finches (Taeniopygia guttata) master the imitation of a song model 80-90 d after hatching and retain it with little change for the rest of their lives. Acquisition and maintenance of this imitation require intact hearing. A previous report showed that male zebra finches deafened as adults start to lose some of the acoustic and temporal features of their song a few weeks after deafening and that by 16 weeks the learned song is severely degraded (Nordeen and Nordeen, 1992). However, this previous study noted no correlation between the age at deafening and the subsequent timing and extent of song loss. We deafened adult male zebra finches ranging in age from 81 d to 6 years. The song of birds deafened at the younger ages (81-175 d) deteriorated severely after a few weeks, and within that age bracket, the older the bird was at deafening, the longer it took for this degradation to occur and the slower the subsequent process of song deterioration. The song of birds deafened at 2 years and older showed little change during the first 51 weeks after deafening but was grossly altered by 100 weeks. We suggest (1) that this age effect could be independent of experience or (2) that each time a bird sings, a little bit of learning-motor engrainment-occurs, adding to memory duration in a cumulative manner.

Expression of polysialylated N-CAM in the central nervous system of adult canaries and its possible relation to function.

Polysialylated neuronal cell adhesion molecule (PSA-N-CAM) is a cell surface molecule associated with neurons that undergo changes in configuration or spatial translocation. In both cases, this molecule is thought to reduce the adhesivity of these cells or of their processes, which can thereby insinuate themselves into the existing parenchyma. We used a monoclonal antibody specific to PSA to offer what we believe is the first account of the distribution of PSA-N-CAM in the adult songbird brain. This antibody stained a diversity of cell classes and processes, as follows: 1) a subset of ventricular zone cells; 2) migrating cells thought to be neuroblasts; 3) a subset of differentiated neurons; 4) some brain surface astrocytes; 5) some tanycytes; 6) the neuropil of some regions; 7) some axonal fibers; and 8) possibly some synapses. Our results demonstrate also, for the first time, the wide distribution of a very numerous population of migrating cells in the telencephalon and the seasonal regulation of PSA-N-CAM expression in a part of the adult brain known to undergo seasonal changes in cell recruitment and function. However, we did not find PSA-N-CAM associated with young migrating cells in the high vocal center (HVC), nor was there PSA-N-CAM in the robust nucleus of the archistriatum (RA), which is known to receive new axonal endings from HVC. In these instances spatial translocation may occur with the assistance of other surface molecules.

Projections of a telencephalic auditory nucleus-field L-in the canary.

Anterograde projections of a telencephalic auditory area - field L of the neostriatum - were traced in canaries, Serinus canarius. Field L was defined as the neostriatal projection of nucleus ovoidalis of the thalamus. Using amino acid autoradiography, two efferent projections of field L and adjacent neostriatum were observed: (1) a projection to the medial and ventral borders of nucleus hyperstriatum ventrale, pars caudale (HVc) and (2) a smaller projection to medial paleostriatum augmentatum (PA). When autoradiographic injection sites included neostriatum postero-ventral to field L, a projection to archistriatum outlining the anterior and ventral borders of the nucleus robustus archistriatalis (RA) resulted. Injection sites that included neostriatum antero-lateral to "L" gave rise to projections to the interior of HVc proper. Above background numbers of silver grains were consistently observed over caudal dorso-lateral portions of nucleus ovoidalis. Following lesion of field L and adjacent neostriatum, argyrophilic degeneration was traced to medial PA and to a shelf of neostriatum underlying HVc. All observed anterograde projections were ipsilateral. Two of the nuclei outlined by neostriatal projections in this study, HVc and RA, have important roles in the motor control of canary song (Nottebohm et al., '76). The development of song is dependent on auditory information. Auditory-vocal neural connections described here may be involved in song learning.