Neuron loss and addition in developing zebra finch song nuclei are independent of auditory experience during song learning

In zebra finches early auditory experience is critical for normal song development. Young males first listen to and memorize a suitable song model and then use auditory feedback from their own vocalizations to mimic that model. During these two phases of vocal learning, song-related brain regions exhibit large, hormone-induced changes in volume and neuron number. Overlap between these neural changes and auditory-based vocal learning suggests that processing and acquiring auditory input may influence cellular processes that determine neuron number in the song system. We addressed this hypothesis by measuring neuron density, nuclear volume, and neuron number within the song system of normal male zebra finches and males deafened prior to song learning (10 days of age). Measures were obtained at 25, 50, 65, and 120 days of age, and included four song nuclei: the hyperstriatum ventralis pars caudalis or higher vocal center (HVc), Area X, the robust nucleus of the archistriatum (RA), and the lateral magnocellular nucleus of the anterior neostriatum (IMAN). In both HVc and Area X, nuclear volume and neuron number increased markedly with age in both normal and deafened birds. The volume of RA also increased with age and was not affected by early deafening. In IMAN, deafening also did not affect the overall age-related loss of neurons, although at 25 days neuron number was slightly less in deafened than in normal birds. We conclude that while the addition and loss of neurons in the developing song system may provide plasticity essential for song learning, these changes do not reflect learning.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Deafening drives cell-type-specific changes to dendritic spines in a sensorimotor nucleus important to learned vocalizations

Hearing loss prevents vocal learning and causes learned vocalizations to deteriorate, but how vocalization-related auditory feedback acts on neural circuits that control vocalization remains poorly understood. We deafened adult zebra finches, which rely on auditory feedback to maintain their learned songs, to test the hypothesis that deafening modifies synapses on neurons in a sensorimotor nucleus important to song production. Longitudinal in vivo imaging revealed that deafening selectively decreased the size and stability of dendritic spines on neurons that provide input to a striatothalamic pathway important to audition-dependent vocal plasticity, and changes in spine size preceded and predicted subsequent vocal degradation. Moreover, electrophysiological recordings from these neurons showed that structural changes were accompanied by functional weakening of both excitatory and inhibitory synapses, increased intrinsic excitability, and changes in spontaneous action potential output. These findings shed light on where and how auditory feedback acts within sensorimotor circuits to shape learned vocalizations.     

Comparative study of long-term reduction of high-frequency song syllables in deafened zebra and Bengalese finches: implications for the role of auditory feedback in song maintenance

Auditory feedback is necessary for adult song maintenance in both oscines and psittacines. Although belonging to phylogenically separated orders, deafened adult oscine Bengalese finches and psittacine budgerigars exhibit similarities in certain aspects of song changes. An interesting question is whether these birds share common mechanisms for song maintenance. Therefore, it is important to compare the effects of deafening on adult song patterns among and within orders. Although zebra and Bengalese finches are closely related oscine species, few studies have performed direct, long-term, quantitative comparisons of their songs after deafening because suitable song characteristics have not been identified. Based on our previous findings for Bengalese finch songs, we analyzed zebra finch songs over 9 months after deafening, focusing on changes in the number of syllables categorized according to fundamental frequencies. Deafened zebra finches demonstrated a gradual but significant decrease in high-frequency syllables and a tendency to increase low-frequency syllables, similar to deafened Bengalese finches. Although this change took longer in zebra finches, the altered proportion of syllables eventually stabilized. Results indicated that adult songs show similar aspects after auditory deprivation, and that neural mechanisms involved in the maintenance of high-frequency song syllables, using auditory feedback, may be present in both finches despite species differences.     

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.     

Basal forebrain cholinergic modulation of auditory activity in the zebra finch song system

The cholinergic basis of auditory "gating" in the sensorimotor nucleus HVc and its efferent target robustus archistriatalis (RA) was investigated in anesthetized zebra finches. Injections of cholinergic agonists carbachol or muscarine into HVc strongly affected discharge rates and diminished auditory responsiveness in both HVc and its target RA, changes toward an awake-like condition. HVc nicotine injections produced similar strong effects in HVc, but weaker and inconsistent effects in RA. Stimulation of basal forebrain (BF) produced an initial transient network shutdown followed by diminished auditory responsiveness in HVc and RA. All stimulation effects were blocked when preceded by HVc injections of nicotinic or muscarinic antagonists. Thus, BF cholinergic modulation of song system auditory activity acting via functionally distinct HVc circuits can contribute to auditory gating. We hypothesize that wakeful BF activity levels block sensory input to motor systems and adaptively change during behavior to allow sensorimotor feedback such as auditory feedback during singing.     

Matters of life and death in the songbird forebrain.

Male zebra finches learn a specific vocal pattern during a restricted period of development. They produce that song in stereotyped form throughout adulthood, and are unable to learn new song patterns. Development of the neural substrate for song learning and behavior is delayed relative to other brain regions, and neural song-control circuits undergo dramatic changes during the period of vocal learning due to both loss of neurons as well as incorporation of newly generated neurons. In contrast, canaries do learn new song patterns in adulthood and modify their vocal repertoires each breeding season. Adult canaries also maintain a large population of dividing cells in the ependymal zone of the telencephalon, and vast numbers of newly generated neurons migrate out to become incorporated into functional circuits and replace older neurons. We review the relationships between cellular and behavioral aspects of song learning in both zebra finches and canaries, as well as the role of gonadal hormones in regulating diverse aspects of the song-control system.     

Matters of life and death in the songbird forebrain

Male zebra finches learn a specific vocal pattern during a restricted period of development. They produce that song in stereotyped form throughout adulthood, and are unable to learn new song patterns. Development of the neural substrate for song learning and behavior is delayed relative to other brain regions, and neural song-control circuits undergo dramatic changes during the period of vocal learning due to both loss of neurons as well as incorporation of newly generated neurons. In contrast, canaries do learn new song patterns in adulthood and modify their vocal repertoires each breeding season. Adult canaries also maintain a large population of dividing cells in the ependymal zone of the telencephalon, and vast numbers of newly generated neurons migrate out to become incorporated into functional circuits and replace older neurons. We review the relationships between cellular and behavioral aspects of song learning in both zebra finches and canaries, as well as the role of gonadal hormones in regulating diverse aspects of the song-control system. © 1992 John Wiley & Sons, Inc.     

Adult bengalese finches (Lonchura striata var. domestica) require real-time auditory feedback to produce normal song syntax

Songbirds develop their songs by imitating songs of adults. For song learning to proceed normally, the bird's hearing must remain intact throughout the song development process. In many species, song learning takes place during one period early in life, and no more new song elements are learned thereafter. In these so-called close-ended learners, it has long been assumed that once song development is complete, audition is no longer necessary to maintain the motor patterns of full song. However, many of these close-ended learners maintain plasticity in overall song organization; the number and the sequence of song elements included in a song of an individual vary from one utterance to another, although no new song elements are added or lost in adulthood. It is conceivable that these species rely on continued auditory feedback to produce normal song syntax. The Bengalese finch is a close-ended learner that produces considerably variable songs as an adult. In the present study, we found that Bengalese finches require real-time auditory feedback for motor control even after song learning is complete; deafening adult finches resulted in development of abnormal song syntax in as little as 5 days. We also found that there was considerable individual variation in the degree of song deterioration after deafening. The neural mechanisms underlying adult song production in different species of songbirds may be more diverse than has been traditionally considered. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 343–356, 1997     

Ontogenetic changes in the pattern of androgen accumulation in song-control nuclei of male zebra finches

The present study examines the development of androgen accumulation in cells of two brain nuclei that are involved in controlling vocal behavior in zebra finches (Poephila guttata). HVc (caudal nucleus of the ventral hyperstriatum) is involved with vocal production in adult birds, and MAN (magnocellular nucleus of the anterior neostriatum) is involved with the initial ability to learn song. In both of these nuclei there is an increase in the proportion of cells that are labeled by systemic injections of tritiated dihydrotestosterone in juvenile male zebra finches during the time when production of song is becoming stereotyped (25-60 days). Within MAN there is an overall loss of cells during this time, such that the absolute number of androgen target cells in MAN remains at a constant level. However, it does not appear to be the case that unlabeled cells are selectively lost from MAN. Rather it appears that both labeled and unlabeled cells are lost, and the absolute number of labeled cells is maintained at a constant level via recruitment of additional labeled cells from the unlabeled population (i.e., some MAN cells that are unlabeled in young birds become labeled in older birds). In line with this hypothesis, there is a large increase in the density of labeling in individual MAN cells, indicating that these cells have an enhanced ability to concentrate androgen. In contrast to the situation in MAN, there is an increase in the overall number of cells within HVc during this time; this increase in total cell number combines with the increased proportion of labeled cells such that the absolute number of androgen target cells in HVc increases threefold. The ability of individual HVc cells to accumulate androgen remains constant. The relationship of these changes in the pattern of androgen accumulation to other aspects of neural and behavioral development related to song in zebra finches are discussed.     

Auditory-vocal cholinergic pathway in the songbird brain

Male zebra finches learn to imitate a tutor's song through auditory and motor learning. The two main song control nuclei in the zebra finch forebrain, the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), receive cholinergic innervation from the ventral paleostriatum (VP) of the basal forebrain which may play a key role in song learning. By injecting neuroanatomical tracers, we found a topographically segregated pathway from nucleus ovoidalis (Ov) to VP that in turn projects in a topographic fashion to HVC and RA. Ov is a major relay in the main ascending auditory pathway. The results suggest that the cholinergic neurons in the VP responsible for song learning are regulated by auditory information from the Ov.     

Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen

Exogenous estrogens, when administered to hatchling female zebra finches, masculinize the morphology and function of their neural vocal control system. The first of two experiments evaluated whether tamoxifen citrate is an antiestrogen in zebra finches, and the second determined whether it would block the masculinization hypothesized to be caused in hatchling males by the males' endogenous estradiol. In the first experiment adult female zebra finches were ovariectomized and injected for 10 days with estradiol benzoate (EB), tamoxifen, EB and tamoxifen combined, or vehicle (control). The dependent variable was oviduct weight. The EB-stimulated growth of the oviduct was blocked by tamoxifen, which had no effects when administered alone. Thus, tamoxifen acts as an antiestrogen in the zebra finch oviduct. In Experiment 2, male and female zebra finches were treated with tamoxifen or vehicle for the first 20 days after hatching. The males were castrated at 20 days. At 60 days we compared the song control regions of experimental and control males and females. In both sexes tamoxifen increased the somatic areas of neurons in RA (robust nucleus of the archistriatum), HVc (caudal nucleus of the ventral hyperstriatum), and MAN (magnocellular nucleus of the anterior neostriatum). Tamoxifen also increased the volumes of HVc, RA, MAN, and Area X in males. Thus, tamoxifen failed to block masculinization of males, but masculinized females and hypermasculinized males. Tamoxifen's hypermasculinization of the male and masculinization of the female song system is paradoxical given that (1) estradiol does not have similar effects on the male song system, and (2) tamoxifen antagonizes the effects of EB in the oviduct.     

Ultrastructural localization of the calcium-binding protein parvalbumin in neurons of the song system of the zebra finch,Poephila guttata

Summary The distribution of parvalbumin (PV) within neurons of the vocal motor nucleus hyperstriatum ventralepars caudalis (HVc) was investigated in the forebrain of adult male zebra finches by means of light and electron microscopy using the indirect immunoperoxidase technique. Parvalbumin-reaction product was located in the amorphous material of perikarya, dendrites and nuclei, and associated to microtubuli, postsynaptic densities and intracellular membranes; it was found in some axons and Gray type-2 boutons, but rarely in type-1 boutons and never in the Golgi apparatus. These observations suggest that parvalbumin may regulate calcium-dependent processes at the postsynaptic membrane and in the cytosol. Furthermore, the partial association of parvalbumin to microtubuli points to an involvement in calcium-dependent tubular functions. Calcium currents and microtubular assembly or transport may be relevant for the known functions of HVc in song learning.     

A sensorimotor area in the songbird brain is required for production of vocalizations in the song learning period of development

Sensory feedback is essential for acquiring and maintaining complex motor behaviors, including birdsong. In zebra finches, auditory feedback reaches the song control circuits primarily through the nucleus interfacialis nidopalii (Nif), which provides excitatory input to HVC (proper name)—a premotor region essential for the production of learned vocalizations. Despite being one of the major inputs to the song control pathway, the role of Nif in generating vocalizations is not well understood. To address this, we transiently inactivated Nif in late juvenile zebra finches. Upon Nif inactivation (in both hemispheres or on one side only), birds went from singing stereotyped zebra finch song to uttering highly variable and unstructured vocalizations resembling sub‐song, an early juvenile song form driven by a basal ganglia circuit. Simultaneously inactivating Nif and LMAN (lateral magnocellular nucleus of the anterior nidopallium), the output nucleus of a basal ganglia circuit, inhibited song production altogether. These results suggest that Nif is required for generating the premotor drive for song. Permanent Nif lesions, in contrast, have only transient effects on vocal production, with song recovering within a day. The sensorimotor nucleus Nif thus produces a premotor drive to the motor pathway that is acutely required for generating learned vocalizations, but once permanently removed, the song system can compensate for its absence. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1213–1225, 2016     

Hemispheric Asymmetry in New Neurons in Adulthood Is Associated with Vocal Learning and Auditory Memory

Many brain regions exhibit lateral differences in structure and function, and also incorporate new neurons in adulthood, thought to function in learning and in the formation of new memories. However, the contribution of new neurons to hemispheric differences in processing is unknown. The present study combines cellular, behavioral, and physiological methods to address whether 1) new neuron incorporation differs between the brain hemispheres, and 2) the degree to which hemispheric lateralization of new neurons correlates with behavioral and physiological measures of learning and memory. The songbird provides a model system for assessing the contribution of new neurons to hemispheric specialization because songbird brain areas for vocal processing are functionally lateralized and receive a continuous influx of new neurons in adulthood. In adult male zebra finches, we quantified new neurons in the caudomedial nidopallium (NCM), a forebrain area involved in discrimination and memory for the complex vocalizations of individual conspecifics. We assessed song learning and recorded neural responses to song in NCM. We found significantly more new neurons labeled in left than in right NCM; moreover, the degree of asymmetry in new neuron numbers was correlated with the quality of song learning and strength of neuronal memory for recently heard songs. In birds with experimentally impaired song quality, the hemispheric difference in new neurons was diminished. These results suggest that new neurons may contribute to an allocation of function between the hemispheres that underlies the learning and processing of complex signals.     

A reafferent and feed-forward model of song syntax generation in the Bengalese finch

Adult Bengalese finches generate a variable song that obeys a distinct and individual syntax. The syntax is gradually lost over a period of days after deafening and is recovered when hearing is restored. We present a spiking neuronal network model of the song syntax generation and its loss, based on the assumption that the syntax is stored in reafferent connections from the auditory to the motor control area. Propagating synfire activity in the HVC codes for individual syllables of the song and priming signals from the auditory network reduce the competition between syllables to allow only those transitions that are permitted by the syntax. Both imprinting of song syntax within HVC and the interaction of the reafferent signal with an efference copy of the motor command are sufficient to explain the gradual loss of syntax in the absence of auditory feedback. The model also reproduces for the first time experimental findings on the influence of altered auditory feedback on the song syntax generation, and predicts song- and species-specific low frequency components in the LFP. This study illustrates how sequential compositionality following a defined syntax can be realized in networks of spiking neurons.     

Factors limiting song acquisition in adult zebra finches

Song learning takes place in two separate or partially overlapping periods, a sensory phase in which a tutor song is memorized and a sensorimotor phase in which a copy of the model is produced. The stage of song development where song becomes stable and stereotyped is called crystallization. Adult birds usually do not learn new song in many species including the zebra finch. However, it is not known whether song crystallization as such or aging impedes adult learning. Exposure to loud noises prevents birds from developing and crystallizing their song, because they cannot control their voice by auditory feedback. Zebra finches even without previous experience of hearing or singing a song failed to learn a song model provided in adulthood. Thus, neither the absence of a tutor song nor the lack of song crystallization enables new song learning in adulthood, but age per se limits the ability or motivation to learn song. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

At the interface of the auditory and vocal motor systems: NIf and its role in vocal processing,production and learning

Communication between auditory and vocal motor nuclei is essential for vocal learning. In songbirds, the nucleus interfacialis of the nidopallium (NIf) is part of a sensorimotor loop, along with auditory nucleus avalanche (Av) and song system nucleus HVC, that links the auditory and song systems. Most of the auditory information comes through this sensorimotor loop, with the projection from NIf to HVC representing the largest single source of auditory information to the song system. In addition to providing the majority of HVC’s auditory input, NIf is also the primary driver of spontaneous activity and premotor-like bursting during sleep in HVC. Like HVC and RA, two nuclei critical for song learning and production, NIf exhibits behavioral-state dependent auditory responses and strong motor bursts that precede song output. NIf also exhibits extended periods of fast gamma oscillations following vocal production. Based on the converging evidence from studies of physiology and functional connectivity it would be reasonable to expect NIf to play an important role in the learning, maintenance, and production of song. Surprisingly, however, lesions of NIf in adult zebra finches have no effect on song production or maintenance. Only the plastic song produced by juvenile zebra finches during the sensorimotor phase of song learning is affected by NIf lesions. In this review, we carefully examine what is known about NIf at the anatomical, physiological, and behavioral levels. We reexamine conclusions drawn from previous studies in the light of our current understanding of the song system, and establish what can be said with certainty about NIf’s involvement in song learning, maintenance, and production. Finally, we review recent theories of song learning integrating possible roles for NIf within these frameworks and suggest possible parallels between NIf and sensorimotor areas that form part of the neural circuitry for speech processing in humans.     

Age effect of deafening on stereotyped song maintenance in adult male bengalese finches Lonchura striata domestica

Birdsong is a complex learned vocal behavior that relies on auditory experience for development.However,it appears that among different species of close-ended songbirds,there are some variations in the necessity of auditory feedback for maintaining stereotyped adult song.In zebra finches,the deterioration of adult songs following deafness depends on the birds' age.It is unknown whether this age effect is a general rule in other avian species as well.Therefore,we chose Bengalese finches,whose songs show more...