Early isolation from conspecific song does not affect the normal developmental decline of N-methyl-D-aspartate receptor binding in an avian song nucleus

Early effects of experience on synaptic reorganization and behavior often involve activation of N-methyl-D -aspartate (NMDA) receptors. We have begun to explore the role of this glutamate-receptor subtype in the development of learned birdsong. Song learning in zebra finches occurs during a restricted period that coincides with extensive synaptic reorganization within neural regions controlling song behavior. In one brain region necessary for song learning, the lateral magnocellular nucleus of the anterior neostriatum (lMAN), NMDA receptor binding is twice as high at the onset of song learning as in adulthood. In the present study, we used quantitative autoradiography with the noncompetitive NMDA antagonist [³H]MK-801 to examine more closely the developmental decline in NMDA receptor binding within lMAN and found that it occurred gradually over the period of song learning and was not associated with a particular stage of the learning process. In addition, early isolation from conspecific song did not affect [³H]MK-801 binding in lMAN at 30, 60, or 80 days. Since behavioral studies confirmed that our isolate rearing conditions extended the sensitive period for song learning, we conclude that the normal developmental decline in overall NMDA receptor binding within lMAN does not terminate the capacity for song learning. Finally, early deafening, which prevents both stages of song learning, also did not affect [³H]MK-801 binding in lMAN at 80 days, indicating that the decline in NMDA receptor binding occurs in the absence of auditory experiences associated with song development. © 1995 John Wiley & Sons, Inc.     

Developmentally restricted synaptic plasticity in a songbird nucleus required for song learning

We provide evidence here of long-term synaptic plasticity in a songbird forebrain area required for song learning, the lateral magnocellular nucleus of the anterior neostriatum (LMAN). Pairing postsynaptic bursts in LMAN principal neurons with stimulation of recurrent collateral synapses had two effects: spike timing- and NMDA receptor-dependent LTP of the recurrent synapses, and LTD of thalamic afferent synapses that were stimulated out of phase with the postsynaptic bursting. Both types of plasticity were restricted to the sensory critical period for song learning, consistent with a role for each in sensory learning. The properties of the observed plasticity are appropriate to establish recurrent circuitry within LMAN that reflects the spatiotemporal pattern of thalamic afferent activity evoked by tutor song. Such circuit organization could represent a tutor song memory suitable for reinforcing particular vocal sequences during sensorimotor learning.     

Circuits,hormones, and learning: Vocal behavior in songbirds

Species-typical vocal patterns subserve species identification and communication for individual organisms. Only a few groups of organisms learn the sounds used for vocal communication, including songbirds, humans, and cetaceans. Vocal learning in songbirds has come to serve as a model system for the study of brain-behavior relationships and neural mechanisms of learning and memory. Songbirds learn specific vocal patterns during a sensitive period of development via a complex assortment of neurobehavioral mechanisms. In many species of songbirds, the production of vocal behavior by adult males is used to defend territories and attract females, and both males and females must perceive vocal patterns and respond to them. In both juveniles and adults, specific types of auditory experience are necessary for initial song learning as well as the maintenance of stable song patterns. External sources of experience such as acoustic cues must be integrated with internal regulatory factors such as hormones, neurotransmitters, and cytokines for vocal patterns to be learned and produced. Thus, vocal behavior in songbirds is a culturally acquired trait that is regulated by multiple intrinsic as well as extrinsic factors. Here, we focus on functional relationships between circuitry and behavior in male songbirds. In that context, we consider in particular the influence of sex hormones on vocal behavior and its underlying circuitry, as well as the regulatory and functional mechanisms suggested by morphologic changes in the neural substrate for song control. We describe new data on the architecture of the song system that suggests strong similarities between the songbird vocal control system and neural circuits for memory, cognition, and use-dependent plasticity in the mammalian brain. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 602–618, 1997     

Acute injections of brain‐derived neurotrophic factor in a vocal premotor nucleus reversibly disrupt adult birdsong stability and trigger syllable deletion

Behavioral variability serves an essential role in motor learning by enabling sensory feedback to select those motor patterns that minimize error. Birds use auditory feedback to learn how to sing, and their songs lose variability and become highly stereotyped, or crystallized, at the end of a sensitive period for sensorimotor learning. The molecular cues that regulate song variability are not well understood. In other systems, neurotrophins, and brain‐derived neurotrophic factor (BDNF) in particular, can mediate various forms of neural plasticity, including sensitive period neural circuit plasticity and activity‐dependent synapse formation, and may also influence learning and memory. Here, we have tested the hypothesis that neurotrophin expression in the robust nucleus of the arcopallium (RA), the telencephalic output controlling song, regulates song variability. BDNF and its receptor trkB are expressed in RA, and BDNF expression in RA appears to be highest in juveniles, when song is most variable and plastic, and synapse density highest. Thus, song variability and synaptic connectivity could be enhanced by augmented expression of BDNF in RA. In support of this idea, we found that BDNF injections into the adult RA induced the re‐expression of juvenile‐like phenotypes, including song variability and an increased synaptic density in RA. Furthermore, BDNF treatment also induced vocal plasticity, characterized by syllable deletions and persistent changes to the song patterns. These results suggest that endogenous BDNF could be a molecular regulator of the song variability essential to vocal plasticity and, ultimately, to song learning. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Acute injections of brain-derived neurotrophic factor in a vocal premotor nucleus reversibly disrupt adult birdsong stability and trigger syllable deletion

Behavioral variability serves an essential role in motor learning by enabling sensory feedback to select those motor patterns that minimize error. Birds use auditory feedback to learn how to sing, and their songs lose variability and become highly stereotyped, or crystallized, at the end of a sensitive period for sensorimotor learning. The molecular cues that regulate song variability are not well understood. In other systems, neurotrophins, and brain-derived neurotrophic factor (BDNF) in particular, can mediate various forms of neural plasticity, including sensitive period neural circuit plasticity and activity-dependent synapse formation, and may also influence learning and memory. Here, we have tested the hypothesis that neurotrophin expression in the robust nucleus of the arcopallium (RA), the telencephalic output controlling song, regulates song variability. BDNF and its receptor trkB are expressed in RA, and BDNF expression in RA appears to be highest in juveniles, when song is most variable and plastic, and synapse density highest. Thus, song variability and synaptic connectivity could be enhanced by augmented expression of BDNF in RA. In support of this idea, we found that BDNF injections into the adult RA induced the re-expression of juvenile-like phenotypes, including song variability and an increased synaptic density in RA. Furthermore, BDNF treatment also induced vocal plasticity, characterized by syllable deletions and persistent changes to the song patterns. These results suggest that endogenous BDNF could be a molecular regulator of the song variability essential to vocal plasticity and, ultimately, to song learning.     

Distribution and developmental change in [3H]MK-801 binding within zebra finch song nuclei

In many songbirds, vocal learning depends upon appropriate auditory experience during a sensitive period that coincides with the formation and reorganization of song-related neural pathways. Because some effects of early sensory experience on neural organization and early learning have been linked to activation of N-methyl-d-aspartate (NMDA) receptors, we measured binding to this receptor within the neural system controlling song behavior in zebra finches. Quantitative autoradiography was used to measure binding of the noncompetitive antagonist [³H]MK-801 (dizocilpine) in the brains of both adult and juvenile male zebra finches, focusing on four telencephalic regions implicated in song learning and production. Overall, the pattern of MK-801 binding in zebra finches was similar to the pattern found in rats (Monaghan and Cotman, 1985, J. Neurosci. 5:2909–2919; Sakurai, Cha, Penney, and Young, 1991, Neuroscience 40:533–543). That is, binding was highest in the telencephalon, intermediate in thalamic regions, and virtually absent from the brain stem and cerebellum. The telencephalic song areas exhibited intermediate levels of binding, and binding in the juveniles was not significantly different from adult levels in most song nuclei. However, in the lateral magnocellular nucleus of the anterior neostriatum (IMAN), binding at 30 days of age was significantly higher than binding in adults. Given the established role of NMDA receptors in other developing neural systems, both their presence in song control nuclei and their developmental regulation within a region implicated in song learning suggest that NMDA receptors play a role in mediating effects of auditory experience on the development of song behavior.     

Sensitive periods and circuits for learned birdsong

Experience influences the development of certain behaviors and their associated neural circuits during a discrete period after birth. Songbirds, with their highly quantifiable vocal output and well-delineated vocal control circuitry, provide an excellent context in which to examine the neural mechanisms regulating sensitive periods for learning. Recent discoveries indicate that auditory input to the vocal control circuitry in songbirds is dynamically modulated and show that neural circuitry previously thought to be used only in plastic juvenile song may also actively maintain stable adult song. These findings provide important clues to how sensitive periods for auditory feedback and vocal plasticity are regulated during song development.     

Comparative approaches to the avian song system

There is extensive diversity among the 4000 species of songbirds in different aspects of song behavior, including the timing of vocal learning, sex patterns of song production, number of songs that are learned (i.e., repertoire size), and seasonality of song behavior. This diversity provides unparalleled opportunities for comparative studies of the relationship between the structure and function of brain regions and song behavior. The comparative approach has been used in two contexts: (a) to test hypotheses about mechanisms of song control, and (b) to study the evolution of the control system in different groups of birds. In the first context, I review studies in which a comparative approach has been used to investigate sex differences in the song system, the relationship between the number of song types a bird sings and the size of the song nuclei, and seasonal plasticity of the song control circuits. In the second context, I discuss whether the vocal control systems of parrots and songbirds were inherited from a common ancestor or independently evolved. I also consider at what stage in the phylogeny of songbirds the hormone-sensitive forebrain circuit found in modern birds first evolved. I conclude by identifying directions for future research in which a comparative approach would be productive. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 517–531, 1997     

SRC3 acetylates calmodulin in the mouse brain to regulate synaptic plasticity and fear learning

Protein acetylation is a reversible posttranslational modification, which is regulated by lysine acetyltransferase (KAT) and lysine deacetyltransferase (KDAC). Although protein acetylation has been shown to regulate synaptic plasticity, this was mainly for histone protein acetylation. The function and regulation of nonhistone protein acetylation in synaptic plasticity and learning remain largely unknown. Calmodulin (CaM), a ubiquitous Ca²⁺ sensor, plays critical roles in synaptic plasticity such as long-term potentiation (LTP). During LTP induction, activation of NMDA receptor triggers Ca²⁺ influx, and the Ca²⁺ binds with CaM and activates calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). In our previous study, we demonstrated that acetylation of CaM was important for synaptic plasticity and fear learning in mice. However, the KAT responsible for CaM acetylation is currently unknown. Here, following an HEK293 cell-based screen of candidate KATs, steroid receptor coactivator 3 (SRC3) is identified as the most active KAT for CaM. We further demonstrate that SRC3 interacts with and acetylates CaM in a Ca²⁺ and NMDA receptor-dependent manner. We also show that pharmacological inhibition or genetic downregulation of SRC3 impairs CaM acetylation, synaptic plasticity, and contextual fear learning in mice. Moreover, the effects of SRC3 inhibition on synaptic plasticity and fear learning could be rescued by 3KQ-CaM, a mutant form of CaM, which mimics acetylation. Together, these observations demonstrate that SRC3 acetylates CaM and regulates synaptic plasticity and learning in mice.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed‐ended and open‐ended learning

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience‐dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open‐ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin‐positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed‐ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin‐positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed‐ended and open‐ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975–994, 2017     

Maturation‐dependent control of vocal temporal plasticity in a songbird

Birdsong is a unique model to address learning mechanisms of the timing control of sequential behaviors, with characteristic temporal structures consisting of serial sequences of brief vocal elements (syllables) and silent intervals (gaps). Understanding the neural mechanisms for plasticity of such sequential behavior should be aided by characterization of its developmental changes. Here, we assessed the level of acute vocal plasticity between young and adult Bengalese finches, and also quantified developmental change in variability of temporal structure. Acute plasticity was tested by delivering aversive noise bursts contingent on duration of a target gap, such that birds could avoid the noise by modifying their song. We found that temporal variability of song features decreased with birds' maturation. Noise‐avoidance experiments demonstrated that maximal changes of gap durations were larger in young that in adult birds. After these young birds matured, the maximal change decreased to a similar level as adults. The variability of these target gaps also decreased as the birds matured. Such parallel changes suggest that the level of acute temporal plasticity could be predicted from ongoing temporal variability. Further, we found that young birds gradually began to stop their song at the target gap and restart from the introductory part of song, whereas adults did not. According to a synaptic chain model for timing sequence generation in premotor nuclei, adult learning would be interpreted as adaptive changes in conduction delays between chain‐to‐chain connections, whereas the learning of young birds could mainly depend on changes of the connections. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 995–1006, 2017     

Neurotensin and neurotensin receptor 1 mRNA expression in song‐control regions changes during development in male zebra finches

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine‐related measures change throughout development in song‐control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song‐control regions of male zebra finches in four stages of the song learning process: pre‐subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre‐subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018     

Vocal Plasticity – are Pied Flycatchers,Ficedula Hypoleuca,Open‐Ended Learners?

In some songbird species, large song repertoires are advantageous in female attraction, whereas song sharing with neighbours may give an advantage in male–male competition. Open‐ended learners, with the ability to memorize new song elements throughout their lives, may learn from territorial neighbours and thus benefit from increasing both repertoire size and song sharing. A distinction needs to be made between true adult song learning, i.e. memorization of novel song elements, and vocal plasticity resulting in changes in the use of previously memorized elements, such as the use of hidden repertoires or increased production of previously rare syllable types. We assessed the ability of adult pied flycatcher Ficedula hypoleuca males to learn previously unheard song elements and to change their song production in response to playback of unfamiliar, conspecific song, emulating a singing neighbour. After a 1‐week playback treatment, three out of 20 subjects had learned foreign song elements, providing evidence from the wild that pied flycatchers are true open‐ended learners. However, the syllable sharing with the playback stimulus repertoires had not changed, and the males’ repertoires had decreased rather than increased. Hence, we did not find support for increased syllable sharing with neighbours or increased repertoire size as functions of adult song learning in pied flycatchers. Because pied flycatcher song seems to serve mainly for mate attraction, copying of attractive syllable types is a possible alternative function of adult song learning in this species.     

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.     

Song learning: the interface between behaviour and neuroethology

The high degree of developmental plasticity displayed by the songs of oscine birds makes them appropriate subjects for research on the etiology and neurobiology of vocal learning. Strong individual differences and learned local dialects are common. The readiness to acquire new songs appears to persist throughout life in some species and is restricted to relatively short sensitive periods in others. Learning can occur with remarkably few exposures to song. Mimicry of other species occurs but, given a choice, there is a tendency to favour conspecific songs. Evidence is presented for two kinds of vocal learning, one 'memory-based', the other 'action-based.' Subsong and 'plastic song' phases of motor development appear to be obligatory steps in the ontogeny of learned songs. A case is made that acquisition and production should be viewed as distinct phenomena with different physiological correlates. Research on behavioural development is closely associated with studies of the physiology of development. The two are mutually synergistic, and the synergism is well displayed in research on song learning in birds. This review of some of the characteristics of avian vocal learning as derived from behavioural studies, indicates lacunae in our knowledge about the ethology of song learning, and suggests how the comparative study of vocal development can pave the way for new insights into the underlying neurobiology.