DARPP-32 distinguishes a subset of adult-born neurons in zebra finch HVC.

Adult male zebra finches (Taeniopygia guttata) continually incorporate adult-born neurons into HVC, a telencephalic brain region necessary for the production of learned song. These neurons express activity-dependent immediate early genes (e.g., zenk and c-fos) following song production, suggesting that these neurons are active during song production. Half of these adult-born HVC neurons (HVC NNs) can be backfilled from the robust nucleus of the arcopallium (RA) and are a part of the vocal motor pathway underlying learned song production, but the other half do not backfill from RA, and they remain to be characterized. Here, we used cell birth-dating, retrograde tract tracing, and immunofluorescence to demonstrate that half of all HVC NNs express the phosphoprotein DARPP-32, a protein associated with dopamine receptor expression. We also demonstrate that DARPP-32+ HVC NNs are contacted by tyrosine hydroxylase immunoreactive fibers, suggesting that they receive catecholaminergic input, have transiently larger nuclei than DARPP-32-neg HVC NNs, and do not backfill from RA. Taken together, these findings help characterize a group of HVC NNs that have no apparent projections to RA and so far have eluded positive identification other than HVC NN status.

A method for exploring adult neurogenesis in the songbird brain.

The avian brain is a valuable model for exploring adult neurogenesis. Here we use immunohistochemical methods to detect cell division and the incorporation of new neurons in the adult zebra finch brain. The nonradioactive, relatively inexpensive thymidine analog bromodeoxyuridine (BrdU) is used to label replicating DNA in dividing cells. The brain is harvested, fixed, and dehydrated before being embedded in polyethylene glycol (PEG), which results in superior histology compared to frozen specimens. After the PEG-embedded brain tissue is sectioned and mounted on slides, standard immunohistochemical procedures are used to detect both BrdU and the neuron-specific marker Hu.

Adult neurogenesis is associated with the maintenance of a stereotyped, learned motor behavior.

Adult neurogenesis is thought to provide neural plasticity used in forming and storing new memories. Here we show a novel relationship between numbers of new neurons and the stability of a previously learned motor pattern. In the adult zebra finch, new projection neurons are added to the nucleus HVC and become part of the motor pathway for producing learned song. However, new song learning occurs only in juveniles and the behavioral impact of adding new neurons to HVC throughout life is unclear. We report that song changes after deafening are inversely correlated with the number of new neurons added to HVC, suggesting that adult neurogenesis in this context may contribute to behavioral stability. More broadly, we propose that new neuron function may depend on the site of integration and can vary as widely as promoting, or restricting, behavioral plasticity.

Anatomical plasticity in the adult zebra finch song system.

In many songbirds, vocal learning-related cellular plasticity was thought to end following a developmental critical period. However, mounting evidence in one such species, the zebra finch, suggests that forms of plasticity common during song learning continue well into adulthood, including a reliance on auditory feedback for song maintenance. This reliance wanes with increasing age, in tandem with age-related increases in fine motor control. We investigated age-related morphological changes in the adult zebra finch song system by focusing on two cortical projection neuron types that 1) share a common efferent target, 2) are known to exhibit morphological and functional change during song learning, and 3) exert opposing influences on song acoustic structure. Neurons in HVC and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) both project to the robust nucleus of the arcopallium (RA). During juvenile song learning and adult song maintenance, HVC promotes song syllable stereotypy, whereas LMAN promotes learning and acoustic variability. After retrograde labeling of these two cell types in adults, there were age-related increases in dendritic arbor in HVC-RA but not LMAN-RA neurons, resulting in an increase in the ratio of HVC-RA:LMAN-RA dendritic arbor. Differential growth of HVC relative to LMAN dendrites may relate to increases in song motor refinement, decreases in the reliance of song on auditory feedback, or both. Despite this differential growth with age, both cell types retain the capacity for experience-dependent growth, as we show here. These results may provide insights into mechanisms that promote and constrain adult vocal plasticity.

Adult neuron addition to the zebra finch song motor pathway correlates with the rate and extent of recovery from botox-induced paralysis of the vocal muscles.

In adult songbirds, neurons are continually incorporated into the telencephalic nucleus HVC (used as a proper name), a premotor region necessary for the production of learned vocalizations. Previous studies have demonstrated that neuron addition to HVC is highest when song is most variable: in juveniles during song learning, in seasonally singing adults during peaks in plasticity that precede the production of new song components, or during seasonal reestablishment of a previously learned song. These findings suggest that neuron addition provides motor flexibility for the transition from a variable song to a target song. Here we test the association between the quality of song structure and HVC neuron addition by experimentally manipulating syringeal muscle control with Botox, which produces a transient partial paralysis. We show that the quality of song structure covaries with new neuron addition to HVC. Both the magnitude of song distortion and the rate of song recovery after syringeal Botox injections were correlated with the number of new neurons incorporated into HVC. We suggest that the quality of song structure is either a cause or consequence of the number of new neurons added to HVC. Birds with naturally high rates of neuron addition may have had the greatest success in recovering song. Alternatively, or in addition, new neuron survival in the song motor pathway may be regulated by the quality of song-generated feedback as song regains its original stereotyped structure. Present results are the first to show a relationship between peripheral muscle control and adult neuron addition to cortical premotor circuits.

The relationship of neurogenesis and growth of brain regions to song learning.

Song learning, maintenance and production require coordinated activity across multiple auditory, sensory-motor, and neuromuscular structures. Telencephalic components of the sensory-motor circuitry are unique to avian species that engage in song learning. The song system shows protracted development that begins prior to hatching but continues well into adulthood. The staggered developmental timetable for construction of the song system provides clues of subsystems involved in specific stages of song learning and maintenance. Progressive events, including neurogenesis and song system growth, as well as regressive events such as apoptosis and synapse elimination, occur during periods of song learning and the transitions between variable and stereotyped song during both development and adulthood. There is clear evidence that gonadal steroids influence the development of song attributes and shape the underlying neural circuitry. Some aspects of song system development are influenced by sensory, motor and social experience, while other aspects of neural development appear to be experience-independent. Although there are species differences in the extent to which song learning continues into adulthood, growing evidence suggests that despite differences in learning trajectories, adult refinement of song motor control and song maintenance can require remarkable behavioral and neural flexibility reminiscent of sensory-motor learning.

Nest of origin predicts adult neuron addition rates in the vocal control system of the zebra finch.

Neurogenesis and neuronal replacement in adulthood represent dramatic forms of plasticity that might serve as a substrate for behavioral flexibility. In songbirds, neurons are continually replaced in HVC (used as a proper name), a pre-motor region necessary for the production of learned vocalizations. There are large individual differences in HVC neuron addition. Some of this variation is probably due to individual differences in adult experience; however, it is also possible that heritability or experience early in development constrains the levels of adult neuron addition. As a step toward addressing the latter two possibilities, we explored the extent to which nest of origin predicts rates of HVC neuron addition in adult male zebra finches. One month after injections of [(3)H]-thymidine to mark dividing cells, neuron addition in HVC was found to co-vary among birds that had been nest mates, even when they were housed in different cages as adults. We also tested whether nest mate co-variation might be due to shared adult auditory experience by measuring neuron addition in nest mate pairs after one member was deafened. There were significant differences in neuron addition between hearing and deaf birds but nest mate relationships persisted. These results suggest that variation in genotype and/or early pre- or postnatal experience can account for a large fraction of adult variation in rates of neuron addition. These results also suggest that a major constraint on neurogenesis and the capacity to adjust rates of neuron addition in response to adult auditory experience is established early in development.

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.

Neuron addition and loss in the song system: regulation and function.

Neurons continue to be produced and replaced throughout life in songbirds. Proliferation in the walls of the lateral ventricles gives rise to neurons that migrate long distances to populate many diverse telencephalic regions, including nuclei dedicated to the perception and production of song, a learned behavior. Many projection neurons are incorporated into the efferent motor pathway for song control. Replacement of these neurons is regulated, in part, by neuron death. Underlying mechanisms include gonadal steroids and BDNF, but are likely to involve other trophic factors as well. The functional significance of neuronal replacement remains unclear. However, recent experiments suggest a link between cell turnover and one or more specific attributes of song learning and production. Several hypotheses are critically examined, including the possibility that neuronal replacement provides motor flexibility to allow for error correction-a capacity needed for juvenile and adult song learning, but also likely to be important for the maintenance of song stereotypy. We highlight important gaps in our knowledge and discuss future directions that may bring us closer to solving the riddle of why neurons are produced and replaced in adulthood.

Vocal control neuron incorporation decreases with age in the adult zebra finch.

In adult male zebra finches, high vocal center (HVC) neurons continuously die and are replaced. Many of these cells are projection neurons that form part of the efferent pathway controlling learned song production. Although it is known that HVC receives new neurons well into adulthood, it is unknown whether this occurs at a constant rate or declines with adult age. We used [3H]thymidine to label new HVC neurons in male zebra finches that were 3-36 months of age. Birds were killed 4 months after 3H injections to measure the long-term incorporation of new HVC neurons. HVC neurons projecting to the robust nucleus of the archistriatum (HVC-RA) were retrogradely labeled with Fluoro-Gold 4 d before death. We found a dramatic age-related decline in the number of 3H-labeled HVC-RA neurons present 4 months after cell birth dating. A similar decline in new HVC neurons was found as soon as 1 month after their formation. These results indicate that the production or early survival of adult-formed neurons decreases with age. HVC volume and total neuron number did not change with bird age, suggesting that the age-related decrease in new neuron addition was balanced by increased survivorship of neurons incorporated previously. Reliance of song structure on auditory feedback also wanes with age. We propose that with aging, fewer new cells are added as the numbers of functionally appropriate cells increase, a process that may be linked to age-related increases in motor program stability.