Song selectivity in the pallial-basal ganglia song circuit of zebra finches raised without tutor song exposure

Acoustic experience critically influences auditory cortical development as well as emergence of highly selective auditory neurons in the songbird sensorimotor circuit. In adult zebra finches, these "song-selective" neurons respond better to the bird's own song (BOS) than to songs of other conspecifics. Birds learn their songs by memorizing a tutor's song and then matching auditory feedback of their voice to the tutor song memory. Song-selective neurons in the pallial-basal ganglia circuit called the anterior forebrain pathway (AFP) reflect the development of BOS. However, during learning, they also respond strongly to tutor song and are compromised in their adult selectivity when birds are prevented from matching BOS to tutor, suggesting that selectivity depends on tutor song learning as well as sensorimotor matching of BOS feedback to the tutor song memory. We examined the contribution of sensory learning of tutor song to song selectivity by recording from AFP neurons in birds reared without exposure to adult conspecifics. We found that AFP neurons in these "isolate" birds had highly tuned responses to isolate BOS. The selectivity was as high, and in the striato-pallidal nucleus Area X, even higher than that in normal birds, due to abnormally weak responsiveness to conspecific song. These results demonstrate that sensory learning of tutor song is not necessary for BOS tuning of AFP neurons. Because isolate birds develop their song via sensorimotor learning, our data further illustrate the importance of individual sensorimotor learning for song selectivity and provide insight into possible functions of song-selective neurons.     

Singing-related activity of identified HVC neurons in the zebra finch

High vocal center (HVC) is part of the premotor pathway necessary for song production and is also a primary source of input to the anterior forebrain pathway (AFP), a basal ganglia-related circuit essential for vocal learning. We have examined the activity of identified HVC neurons of zebra finches during singing. Antidromic activation was used to identify three classes of HVC cells: neurons projecting to the premotor nucleus RA, neurons projecting to area X in the AFP, and putative HVC interneurons. HVC interneurons are active throughout the song and display tonic patterns of activity. Projection neurons exhibit highly phasic stereotyped firing patterns. X-projecting (HVC((X))) neurons burst zero to four times per motif, whereas RA-projecting neurons burst extremely sparsely--at most once per motif. The bursts of HVC projection neurons are tightly locked to the song and typically have a jitter of <1 ms. Population activity of interneurons, but not projection neurons, was significantly correlated with syllable patterns. Consistent with the idea that HVC codes for the temporal order in the song rather than for sound, the vocal dynamics and neural dynamics in HVC occur on different and uncorrelated time scales. We test whether HVC((X)) neurons are auditory sensitive during singing. We recorded the activity of these neurons in juvenile birds during singing and found that firing patterns of these neurons are not altered by distorted auditory feedback, which is known to disrupt learning or to cause degradation of song already learned.     

HVC neural sleep activity increases with development and parallels nightly changes in song behavior

Sleep abnormalities are coexpressed with human communication disorders. Recent data from the birdsong system, the best model for human speech, indicate that sleep has a critical role in vocal learning. To understand the neural mechanisms that underlie behavioral changes during sleep, we recorded sleep activity in the song control area HVC longitudinally during song development in zebra finches. We focused on the sensorimotor phase of song learning, when the finch shapes his song behavior toward a learned tutor song model. Direct comparison of sleep activity in adults and juveniles revealed that the juvenile HVC has a lower spike rate and longer silent periods than the adult. Within individual finches, sleep silent periods decreased and spike rate increased with age. We next systematically compared neural sleep activity and song behavior. We now report that spike rate during sleep was significantly correlated with overnight changes in song behavior. Collectively, these data indicate that sleep activity in the vocal control area HVC increases with age and may affect song behavior.     

Developmental modulation of the temporal relationship between brain and behavior

Humans and songbirds shape learned vocalizations during a sensorimotor sensitive period or "babbling" phase. The brain mechanisms that underlie the shaping of vocalizations by sensory feedback are not known. We examined song behavior and brain activity in zebra finches during singing as they actively shaped their song toward a tutor model. We now show that the temporal relationship of behavior and activity in the premotor area HVC changes with the development of song behavior. During sensorimotor learning, HVC bursting activity both preceded and followed learned vocalizations by hundreds of milliseconds. Correspondingly, the duration of bursts that occurred during ongoing song motif behavior was prolonged in juveniles, as compared with adults, and was inversely correlated with song maturation. Multielectrode single-unit recording in juveniles revealed that single fast-spiking neurons were active both before and after vocalization. These same neurons responded to auditory stimuli. Collectively, these data indicate that a key aspect of sensory critical periods--prolonged bursting--also applies to sensorimotor development. In addition, prolonged motor discharge and sensory input coincide in single neurons of the developing song system, providing the necessary cellular elements for sensorimotor shaping through activity-dependent mechanisms.     

Synaptic interactions underlying song-selectivity in the avian nucleus HVC revealed by dual intracellular recordings

Stimulus-dependent synaptic interactions underlying selective sensory representations in neural circuits specialized for sensory processing and sensorimotor integration remain poorly understood. The songbird telencephalic nucleus HVC is a sensorimotor area essential to learned vocal control with one projection neuron (PN) type (HVC(RA)) innervating a song premotor pathway, another PN (HVC(X)) innervating a basal ganglia pathway essential to vocal plasticity, and interneurons (HVC(Int)). Playback of the bird's own song (BOS), but not other songs, evokes action potential bursts from both PNs, but HVC(RA) and HVC(X) display distinct BOS-evoked subthreshold responses. To characterize synaptic interactions underlying HVC's BOS-selective responses and assess stimulus-evoked changes in functional interactions between HVC neurons, we made simultaneous in vivo intracellular recordings from various HVC neuron pairs in urethan-anesthetized zebra finches. Spike-triggered averaging revealed that all HVC neuron types receive common excitation and that the onset of this excitation occurs during a narrower time window in projection neurons during BOS playback. To distinguish local from extrinsic contributions to HVC subthreshold response patterns, we inactivated the HVC local circuit with GABA or occluded inhibition in single HVC(X) cells. After either treatment, BOS-evoked responses in HVC(X) neurons became purely depolarizing and subthreshold responses of HVC(X) and HVC(RA) cells became remarkably similar to one another while retaining BOS selectivity. Therefore both PN types receive a common extrinsic source of BOS-selective excitation, and local inhibition specifically alters processing of auditory information in HVC(X) cells. In HVC, excitatory and inhibitory synaptic interactions are recruited in a stimulus-dependent fashion, affecting auditory representations of the BOS locally and in other song nuclei important to song learning and production.     

Noradrenergic and GABA B receptor activation differentially modulate inputs to the premotor nucleus RA in zebra finches

Neuromodulators can rapidly modify neural circuits, altering behavior. Songbirds provide an excellent system for studying the role of neuromodulation in modifying circuits that underlie behavior because song learning and production are mediated by a discrete set of interconnected nuclei. We examined the neuromodulatory effects of noradrenergic and GABA B receptor activation on synaptic inputs to the premotor robust nucleus of the arcopallium (RA) in zebra finches using whole cell voltage-clamp recording in vitro. In adults, norepinephrine strongly reduced input from the lateral magnocellular nucleus of the anterior nidopallium (LMAN) but only slightly reduced the input from nucleus HVC (proper name), the excitatory input from axon collaterals of other RA neurons, and input from GABAergic interneurons. The effect of norepinephrine was mimicked by the alpha2 adrenoceptor agonist UK14,304 and blocked by the alpha2 antagonist yohimbine. Conversely, the GABA B receptor agonist baclofen strongly decreased HVC, collateral, and GABAergic inputs to RA neurons while causing little reduction in the LMAN input. In juveniles undergoing song learning, norepinephrine reduced the LMAN input, caused only a small reduction in the HVC input, and greatly reduced the collateral and GABAergic inputs. Baclofen caused similar results in juvenile and adult birds, reducing HVC, collateral, and GABAergic inputs significantly more than the LMAN input. Significant increases in paired-pulse ratio accompanied all reductions in synaptic transmission, suggesting a presynaptic locus. The reduction in the LMAN input by norepinephrine may be important for mediating changes in song elicited by different social contexts and is well-placed to play a role in song learning.     

An associational model of birdsong sensorimotor learning I. Efference copy and the learning of song syllables

Birdsong learning provides an ideal model system for studying temporally complex motor behavior. Guided by the well-characterized functional anatomy of the song system, we have constructed a computational model of the sensorimotor phase of song learning. Our model uses simple Hebbian and reinforcement learning rules and demonstrates the plausibility of a detailed set of hypotheses concerning sensory-motor interactions during song learning. The model focuses on the motor nuclei HVc and robust nucleus of the archistriatum (RA) of zebra finches and incorporates the long-standing hypothesis that a series of song nuclei, the Anterior Forebrain Pathway (AFP), plays an important role in comparing the bird's own vocalizations with a previously memorized song, or "template." This "AFP comparison hypothesis" is challenged by the significant delay that would be experienced by presumptive auditory feedback signals processed in the AFP. We propose that the AFP does not directly evaluate auditory feedback, but instead, receives an internally generated prediction of the feedback signal corresponding to each vocal gesture, or song "syllable." This prediction, or "efference copy," is learned in HVc by associating premotor activity in RA-projecting HVc neurons with the resulting auditory feedback registered within AFP-projecting HVc neurons. We also demonstrate how negative feedback "adaptation" can be used to separate sensory and motor signals within HVc. The model predicts that motor signals recorded in the AFP during singing carry sensory information and that the primary role for auditory feedback during song learning is to maintain an accurate efference copy. The simplicity of the model suggests that associational efference copy learning may be a common strategy for overcoming feedback delay during sensorimotor learning.     

TrkB-like immunoreactivity in the song system of developing zebra finches

The neural song system in zebra finches develops for approximately the first 2 months after hatching. During that time, male-biased sexual dimorphisms emerge in the volume of song control nuclei as well as in the number and size of neurons within them. Brain derived neurotrophic factor (BDNF) has been documented in song control nuclei at various stages of development. Its high affinity receptor (tyrosine kinase B; trkB) is also in the song system, at least at around I month of age. The present study was designed to more completely describe the timing and potential location of BDNF action by investigating trkB expression during sexual differentiation of the song control nuclei. The pattern of immunoreactivity to a trkB antibody was examined in male and female zebra finches at post-hatching days 3-60. Labeling in somata and neuropil appeared to define the telencephalic components of the motor pathway (high vocal center and robust nucleus of the archistriatum) for song production in males from days 30 to 60, and in females on days 45 and 60 (high vocal center). These results are consistent with the hypothesis that the receptor, and its ligand BDNF, play a role in processes related to song learning in both sexes, including perhaps the motor component exhibited by developing males.     

Androgens and isolation from adult tutors differentially affect the development of songbird neurons critical to vocal plasticity

Song learning in oscine birds occurs during a juvenile sensitive period. One idea is that this sensitive period is regulated by changes in the electrophysiological properties of neurons in the telencephalic song nucleus lateral magnocellular nucleus of the anterior neostriatum (LMAN), a structure critical for song development but not adult singing. A corollary of this idea is that manipulations affecting the pace and quality of song learning will concomitantly affect the development of LMAN's electrophysiological properties. Manipulations known to affect song development include treating juvenile male zebra finches with exogenous androgens, which results in abnormally truncated adult songs, and isolation of the juvenile from adult tutors and their songs, which extends the sensitive period for song learning. Previously, we showed that synaptic transmission in LMAN changes over normal song development and that these changes are accelerated or retarded, respectively, by androgen treatment and isolation from an adult tutor. The intrinsic properties of LMAN neurons afford another potential target for regulation by steroid hormones and experience of adult tutors. Indeed previous studies showed that the capacity for LMAN neurons to fire action potentials in bursts, due to a low-threshold calcium spike, and the width of single action potentials in LMAN, wane over development. Here we analyzed these and other intrinsic electrophysiological features of LMAN neurons over normal development, then tested whether either early androgen treatment or isolating juveniles from adult tutors affected the timing of these changes. The present study shows that androgen but not isolation treatment alters the developmental time at which LMAN neurons progress from the bursting to nonbursting phenotype. In addition, other intrinsic properties, including the half-height spike width and the magnitude of the spike afterhyperpolarization (AHP), were found to change markedly over development but only changes to the AHP were androgen sensitive. Interestingly of all of the synaptic and intrinsic electrophysiological properties in LMAN studied to date, only the half-height spike width continues to change in the late juvenile stages of song learning. Furthermore raising juveniles in isolation from an adult tutor transiently delays the maturation of this property. The present results underscore that beyond their effects on LMAN's synaptic properties, both androgens and adult tutor experience are potent and selective regulators of the intrinsic properties of LMAN neurons.     

Endocannabinoids mediate synaptic plasticity at glutamatergic synapses on spiny neurons within a basal ganglia nucleus necessary for song learning

Activation of type 1 cannabinoid receptors (CB(1)R) in many central nervous system structures induces both short- and long-term changes in synaptic transmission. Within mammalian striatum, endocannabinoids (eCB) are one of several mechanisms that induce synaptic plasticity at glutamatergic terminals onto medium spiny neurons. Striatal synaptic plasticity may contribute a critical component of adaptive motor coordination and procedural learning. Songbirds are advantageous for studying the neural mechanisms of motor learning because they possess a neural pathway necessary for song learning and adult song plasticity that includes a striato-pallidal nucleus, area X (homologous to a portion of mammalian basal ganglia). Recent findings suggest that eCBs contribute to vocal development. For example, dense CB(1)R expression in song control nuclei peaks around the closure of the sensori-motor integration phase of song development. Also, systemic administration of a CB(1)R agonist during vocal development impairs song learning. Here we test whether activation of CB(1)R alters excitatory synaptic input on spiny neurons in area X of adult male zebra finches. Application of the CB(1)R agonist WIN55212-2 decreased excitatory postsynaptic current (EPSC) amplitude; that decrease was blocked by the CB(1)R antagonist AM251. Guided by eCB experiments in mammalian striatum, we tested and verified that at least two mechanisms indirectly activate CB(1)Rs through eCBs in area X. First, activation of group I metabotropic glutamate receptors with the agonist 3,5-dihydroxyphenylglycine (DHPG) induced a CB(1)R-mediated reduction in EPSC amplitude. Second, we observed that a 10 s postsynaptic depolarization induced a calcium-mediated, eCB-dependent decrease in synaptic strength that resisted rescue with late CB(1)R blockade. Together, these results show that eCB modulation occurs at inputs to area X spiny neurons and could influence motor learning and production.     

GABA immunoreactivity in auditory and song control brain areas of zebra finches

Inhibitory transmission is critical to sensory and motor processing and is believed to play a role in experience-dependent plasticity. The main inhibitory neurotransmitter in vertebrates, GABA, has been implicated in both sensory and motor aspects of vocalizations in songbirds. To understand the role of GABAergic mechanisms in vocal communication, GABAergic elements must be characterized fully. Hence, we investigated GABA immunohistochemistry in the zebra finch brain, emphasizing auditory areas and song control nuclei. Several nuclei of the ascending auditory pathway showed a moderate to high density of GABAergic neurons including the cochlear nuclei, nucleus laminaris, superior olivary nucleus, mesencephalic nucleus lateralis pars dorsalis, and nucleus ovoidalis. Telencephalic auditory areas, including field L subfields L1, L2a and L3, as well as the caudomedial nidopallium (NCM) and mesopallium (CMM), contained GABAergic cells at particularly high densities. Considerable GABA labeling was also seen in the shelf area of caudodorsal nidopallium, and the cup area in the arcopallium, as well as in area X, the lateral magnocellular nucleus of the anterior nidopallium, the robust nucleus of the arcopallium and nidopallial nucleus HVC. GABAergic cells were typically small, most likely local inhibitory interneurons, although large GABA-positive cells that were sparsely distributed were also identified. GABA-positive neurites and puncta were identified in most nuclei of the ascending auditory pathway and in song control nuclei. Our data are in accordance with a prominent role of GABAergic mechanisms in regulating the neural circuits involved in song perceptual processing, motor production, and vocal learning in songbirds.     

Long-range inhibition within the zebra finch song nucleus RA can coordinate the firing of multiple projection neurons.

The zebra finch forebrain song control nucleus RA (robust nucleus of the archistriatum) generates a phasic and temporally precise neural signal that drives vocal and respiratory motoneurons during singing. RA's output during singing predicts individual notes, even though afferent drive to RA from the song nucleus HVc is more tonic, and predicts song syllables, independent of the particular notes that comprise the syllable. Therefore RA's intrinsic circuitry transforms neural activity from HVc into a highly precise premotor output. To understand how RA's intrinsic circuitry effects this transformation, we characterized RA interneurons and projection neurons using intracellular recordings in brain slices. RA interneurons fired fast action potentials with steep current-frequency relationships and had small somata with thin aspinous processes that extended throughout large portions of the nucleus; the similarity of their fine processes to those labeled with a glutamic acid decarboxylase (GAD) antibody strongly suggests that these interneurons are GABAergic. Electrical stimulation revealed that RA interneurons receive excitatory inputs from RA's afferents, the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and HVc, and from local axon collaterals of RA projection neurons. To map the functional connections that RA interneurons make onto RA projection neurons, we focally uncaged glutamate, revealing long-range inhibitory connections in RA. Thus these interneurons provide fast feed-forward and feedback inhibition to RA projection neurons and could help create the phasic pattern of bursts and pauses that characterizes RA output during singing. Furthermore, selectively activating the inhibitory network phase locks the firing of otherwise unconnected pairs of projection neurons, suggesting that local inhibition could coordinate RA output during singing.     

黄眉鹀上纹状体腹侧尾核的两条发声控制通路的探讨

用ＨＲＰ顺、逆行追踪方法，研究黄眉（ＥｍｂｅｒｉｚａＣｈｒｙｓｏｐｈｒｙｓ）端脑新纹状体前部大细胞核外侧部（ＩＭＡＮ）及嗅叶Ｘ区的纤维联系；并将ＨＲＰ溶液分别微电泳入端脑上纹状体腹侧尾核（ＨＶｃ）及古纹状体粗核（ＲＡ），观察其传出投射。结果发现：（１）ＩＭＡＮ接受丘脑背外侧核内侧部的传入投射，由ＩＭＡＮ发出的纤维投射至ＲＡ；（２）Ｘ区接受ＨＶｃ的传入投射，并发出纤维投射至丘脑背外侧核的内侧部；（３）ＨＶｃ发出两束纤维，分别投射至ＲＡ及Ｘ区；（４）ＲＡ也发出两束纤维，分别投射至中脑背内侧核及延髓舌下神经核气管鸣管部。因此ＨＶｃ可能通过两条路径控制发声行为：一条是ＨＶｃ直接投射至ＲＡ，再由ＲＡ支配延髓舌下神经核气管鸣管部的直接通路；另一条是ＨＶｃ→嗅叶Ｘ区→丘脑背外侧核内侧部→ＩＭＡＮ→ＲＡ的间接通路。后者可能参与发声学习和记忆等高级机能。     

Regulation of learned vocal behavior by an auditory motor cortical nucleus in juvenile zebra finches

In the process of song learning, songbirds such as the zebra finch shape their initial soft and poorly formed vocalizations (subsong) first into variable plastic songs with a discernable recurring motif and then into highly stereotyped adult songs. A premotor brain area critically involved in plastic and adult song production is the cortical nucleus HVC. One of HVC's primary afferents, the nucleus interface of the nidopallium (NIf), provides a significant source of auditory input to HVC. However, the premotor involvement of NIf has not been extensively studied yet. Here we report that brief and reversible pharmacological inactivation of NIf in juvenile birds leads to transient degradation of plastic song toward subsong, as revealed by spectral and temporal song features. No such song degradation is seen following NIf inactivation in adults. However, in both juveniles and adults NIf inactivation leads to a transient decrease in song stereotypy. Our findings reveal a contribution of NIf to song production in juveniles that agrees with its known role in adults in mediating thalamic drive to downstream vocal motor areas during sleep.     

Sex differences in the developmental changes of GABAergic neurons in zebra finch song control nuclei

Small neurons in a sexually dimorphic nucleus of the zebra finch, the robust nucleus of the archistriatum (RA), were immunoreactive for -aminobutyric acid (GABA). This dimorphism arose from the sex differences in the development of the RA neurons. We examined whether neurons with GABA-like immunoreactivity (GABA-LI) undergo the same developmental changes as the cresyl-violet-stained neurons in the RA. Somata with GABA-LI from male finches became larger during development, while somata with GABA-LI from female finches became smaller, at a rate similar to that in the total population of RA neurons. However, there were marked sex differences in the development of the number of RA neurons with GABA-LI. In the female, neurons with GABA-LI were lost at a rate which was similar to the rate of loss of the total population of RA neurons. On the other hand, in the male, the number of neurons with GABA-LI increased transiently during the sensitive period of song learning, while the total number of RA neurons did not change. This transient increase may be related to the fact that only male birds learn their song during a sensitive period.     

An associational model of birdsong sensorimotor learning II. Temporal hierarchies and the learning of song sequence

Understanding the neural mechanisms underlying serially ordered behavior is a fundamental problem in motor learning. We present a computational model of sensorimotor learning in songbirds that is constrained by the known functional anatomy of the song circuit. The model subsumes our companion model for learning individual song "syllables" and relies on the same underlying assumptions. The extended model addresses the problem of learning to produce syllables in the correct sequence. Central to our approach is the hypothesis that the Anterior Forebrain Pathway (AFP) produces signals related to the comparison of the bird's own vocalizations and a previously memorized "template." This "AFP comparison hypothesis" is challenged by the lack of a direct projection from the AFP to the song nucleus HVc, a candidate site for the generator of song sequence. We propose that sequence generation in HVc results from an associative chain of motor and sensory representations (motor --> sensory --> next motor. ) encoded within the two known populations of HVc projection neurons. The sensory link in the chain is provided, not by auditory feedback, but by a centrally generated efference copy that serves as an internal prediction of this feedback. The use of efference copy as a substitute for the sensory signal explains the ability of adult birds to produce normal song immediately after deafening. We also predict that the AFP guides sequence learning by biasing motor activity in nucleus RA, the premotor nucleus downstream of HVc. Associative learning then remaps the output of the HVc sequence generator. By altering the motor pathway in RA, the AFP alters the correspondence between HVc motor commands and the resulting sensory feedback and triggers renewed efference copy learning in HVc. Thus, auditory feedback-mediated efference copy learning provides an indirect pathway by which the AFP can influence sequence generation in HVc. The model makes predictions concerning the role played by specific neural populations during the sensorimotor phase of song learning and demonstrates how simple rules of associational plasticity can contribute to the learning of a complex behavior on multiple time scales.     

State and neuronal class-dependent reconfiguration in the avian song system

Sensory systems may adapt to behavioral requirements through state-dependent changes. In the forebrain song-system nucleus HVc of zebra finches, state-dependent auditory responses have been described in multiunit recordings. Here we report on behavioral state-dependent changes in the activity of distinct HVc neuronal classes. HVc projection neurons were identified by electrically stimulating HVc's target nuclei, the robust nucleus of the archistriatum and Area X, in anesthetized zebra finches. Projection neurons and two classes of putative interneurons could be distinguished on the basis of extracellular spike waveforms, with the first two factors of a principal components analysis accounting for 81% of the variance in spike morphometric values. Spike width was the best single variable for distinguishing among the neuronal classes. Putative interneurons had much higher firing rates spontaneously and in response to song than did projection neurons, which had extremely low spontaneous rates and phasic responses to song. Recordings from HVc in behaving animals were dominated by the two classes of putative interneurons. Both classes showed strong, selective, and temporally similar auditory responses during sleep, but only one class of interneurons reliably maintained auditory responses on waking. These responses were weaker and less selective than those seen during sleep. The observation that HVc auditory responsiveness in awake zebra finches is restricted to some classes of neurons may help explain prior multiunit results that suggested nearly complete suppression of HVc auditory responses in awake birds. We propose that the heterogeneous effects of behavioral state on distinct subpopulations of HVc neurons allow HVc to participate in multiple roles during song production, conspecific song recognition, and possibly memory consolidation during sleep.