Adult Zebra Finches Research Articles

Effect of testosterone on electrophysiological properties of RA projection neurons in adult female zebra finches (Taeniopygia guttata)

Effect of testosterone on electrophysiological properties of RA projection neurons in adult female zebra finches (Taeniopygia guttata)

The expression of DARPP-32 in adult male zebra finches (Taenopygia guttata).

Although the catecholaminergic circuitry in the zebra finch brain has been well studied, there is little information regarding the postsynaptic targets of dopamine. To answer this question, we looked at overall patterns of immunoreactivity for DARPP-32 (a dopamine and cAMP-regulated phosphoprotein, present mostly in dopaminoceptive neurons) in adult male zebra finches. Our results demonstrated that as in mammals and other avian species, DARPP-32 expression was highest in both medial and lateral striatum. Interestingly, a specific pattern of immunoreactivity was observed in the song control system, with 'core' song control regions, that is, LMANcore (lateral magnocellular nucleus of the anterior nidopallium), RA (nucleus robustus arcopallialis) and HVC being less immunoreactive for DARPP-32 than 'shell' areas such as LMANshell, RAcup, AId (intermediate arcopallium) and HVCshelf. Our results suggest that whereas dopamine may modulate the shell pathways at various levels of the AFP, dopaminergic modulation of the core pathway occurs mainly through Area X, a basal ganglia nucleus. Further, secondary sensory cortices including the perientopallial belt, Fields L1 and L3 had higher DARPP-32-immunoreactivity than primary sensory cortical areas such as the pallial basolateral nucleus, entopallium proper and Field L2, corresponding to somatosensory, visual and auditory systems, respectively. We also found DARPP-32-rich axon terminals surrounding dopaminergic neurons in the ventral tegmental area-substantia nigra complex which in turn project to the striatum, suggesting that there may be a reciprocal modulation between these regions. Overall, DARPP-32 expression appears to be higher in areas involved in integrating sensory information, which further supports the role of this protein as a molecular integrator of different signal processing pathways.

Sexual dimorphism of inhibitory synaptic transmission in RA projection neurons of songbirds

The song control system in the brain of songbirds is important for the production and acquisition of song and exhibits some of the largest neural sex differences observed in vertebrates. The robust nucleus of the arcopallium (RA) is a premotor nucleus, playing a key role in controlling singing. RA projection neurons (PNs) receives denser synapse inputs including excitatory in males than in females. However, the inhibitory synaptic transmission in the RA has not been reported. In the present study, using whole-cell voltage-clamp recording, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of the males and females were recorded. The average frequency and amplitude of sIPSCs/mIPSCs in males were higher than females. These results demonstrate the sexually dimorphic of the inhibitory synaptic transmission in the RA PNs and the RA PNs in males receive more inhibitory synaptic transmission. These findings contribute to further illuminate the neural mechanisms under the sexually dimorphism song production of adult zebra finches.

Cannabidiol improves vocal learning-dependent recovery from, and reduces magnitude of deficits following, damage to a cortical-like brain region in a songbird pre-clinical animal model

Cannabidiol (CBD), a non-euphorigenic compound derived from Cannabis, shows promise for improving recovery following cerebral ischemia and has recently been shown effective for the treatment of childhood seizures caused by Dravet and Lennox-Gastaut syndromes. Given evidence for activity to mitigate effects of CNS insult and dysfunction, we considered the possibility that CBD may also protect and improve functional recovery of a complex learned behavior. To test this hypothesis, we have applied a songbird, the adult male zebra finch, as a novel pre-clinical animal model. Their learned vocalizations were temporarily disrupted with bilateral microlesions of HVC (used as a proper name) a pre-vocal motor cortical-like brain region that drives song. These microlesions destroy about 10% of HVC, and temporarily impair song production, syntax and phonology for about seven days. Recovery requires sensorimotor learning as it depends upon auditory feedback. Four CBD doses (0, 1, 10 and 100 mg/kg) within three surgery conditions (microlesion, no-microlesion, sham-microlesion) were evaluated (n = 5–6). Birds were recorded over 20 days: three baseline; six pre-microlesion drug treatment days and; 11 post-microlesion treatment and recovery days. Results indicate 10 and 100 mg/kg CBD effectively reduced the time required to recover vocal phonology and syntax. In the case of phonology, the magnitude of microlesion-related disruptions were also reduced. These results suggest CBD holds promise to improve functional recovery of complex learned behaviors following brain injury, and represent establishment of an important new animal model to screen drugs for efficacy to improve vocal recovery.

Developmental modulation and predictability of age-dependent vocal plasticity in adult zebra finches

Predicting the nature of behavioral plasticity can provide insight into mechanisms of behavioral expression and control. Songbirds like the zebra finch rely on vocal signals for communication, and the performance of these signals demonstrate considerable plasticity over development. Traditionally, these signals were thought to be fixed in adulthood, but recent studies have revealed significant age-dependent changes to spectral and temporal features of song in adult songbirds. A number of age-dependent changes to song resemble acute changes to adult song performance across social contexts (e.g., when an adult male sings to a female relative to when he sings in isolation). The ability of variation in social context-dependent changes to predict variation in age-dependent plasticity would suggest shared mechanisms, but little is known about this predictability. In addition, although developmental experiences can shape adult plasticity, little is known about the extent to which social interactions during development affect age-dependent change to adult song. To this end, we systematically analyzed age- and context-dependent changes to adult zebra finch song, and then examined the degree to which age-dependent changes varied across birds that were social or non-socially tutored birds and to which social context-dependent changes predicted age-dependent changes. Non-socially tutored birds showed more dramatic changes to the broad structure of their motif over time than socially tutored birds, but non-socially and socially tutored birds did not differ in the extent of changes to various spectral and temporal features of song. Overall, we found that adult zebra finches produced longer and more spectrally stereotyped songs when they were older than when they were younger. Moreover, regardless of developmental tutoring, individual variation in age-dependent changes to song bout duration and syllable repetition were predicted by variation in social context-dependent changes to these features. These data indicate that social experiences during development can shape some aspects of adult plasticity and that acute context-dependent and long-term age-dependent changes to some song features could be mediated by modifications within similar neural substrates.

Beyond Critical Period Learning: Striatal FoxP2 Affects the Active Maintenance of Learned Vocalizations in Adulthood.

In humans, mutations in the transcription factor forkhead box P2 (FOXP2) result in language disorders associated with altered striatal structure. Like speech, birdsong is learned through social interactions during maturational critical periods, and it relies on auditory feedback during initial learning and on-going maintenance. Hearing loss causes learned vocalizations to deteriorate in adult humans and songbirds. In the adult songbird brain, most FoxP2-enriched regions (e.g., cortex, thalamus) show a static expression level, but in the striatal song control nucleus, area X, FoxP2 is regulated by singing and social context: when juveniles and adults sing alone, its levels drop, and songs are more variable. When males sing to females, FoxP2 levels remain high, and songs are relatively stable: this “on-line” regulation implicates FoxP2 in ongoing vocal processes, but its role in the auditory-based maintenance of learned vocalization has not been examined. To test this, we overexpressed FoxP2 in both hearing and deafened adult zebra finches and assessed effects on song sung alone versus songs directed to females. In intact birds singing alone, no changes were detected between songs of males expressing FoxP2 or a GFP construct in area X, consistent with the marked stability of mature song in this species. In contrast, songs of males overexpressing FoxP2 became more variable and were less preferable to females, unlike responses to songs of GFP-expressing control males. In deafened birds, song deteriorated more rapidly following FoxP2 overexpression relative to GFP controls. Together, these experiments suggest that behavior-driven FoxP2 expression and auditory feedback interact to precisely maintain learned vocalizations.

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution.

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies.

Sensory feedback independent pre-song vocalizations correlate with time to song initiation.

The song of the adult male zebra finch is a well-studied example of a learned motor sequence. Song bouts begin with a variable number of introductory notes (INs) before actual song production. Previous studies have shown that INs progress from a variable initial state to a stereotyped final state before each song. This progression is thought to represent motor preparation, but the underlying mechanisms remain poorly understood. Here, we assessed the role of sensory feedback in the progression of INs to song. We found that the mean number of INs before song and the progression of INs to song were not affected by removal of two sensory feedback pathways (auditory or proprioceptive). In both feedback-intact and feedback-deprived birds, the presence of calls (other non-song vocalizations), just before the first IN, was correlated with fewer INs before song and an initial state closer to song. Finally, the initial IN state correlated with the time to song initiation. Overall, these results show that INs do not require real-time sensory feedback for progression to song. Rather, our results suggest that changes in IN features and their transition to song are controlled by internal neural processes, possibly involved in getting the brain ready to initiate a learned movement sequence.

Differential developmental changes in cortical representations of auditory-vocal stimuli in songbirds

Procedural skill learning requires iterative comparisons between feedback of self-generated motor output and a goal sensorimotor pattern. In juvenile songbirds, neural representations of both self-generated behaviors (each bird's own immature song) and the goal motor pattern (each bird's adult tutor song) are essential for vocal learning, yet little is known about how these behaviorally relevant stimuli are encoded. We made extracellular recordings during song playback in anesthetized juvenile and adult zebra finches ( Taeniopygia guttata) in adjacent cortical regions RA (robust nucleus of the arcopallium), AId (dorsal intermediate arcopallium), and RA cup, each of which is well situated to integrate auditory-vocal information: RA is a motor cortical region that drives vocal output, AId is an adjoining cortical region whose projections converge with basal ganglia loops for song learning in the dorsal thalamus, and RA cup surrounds RA and receives inputs from primary and secondary auditory cortex. We found strong developmental differences in neural selectivity within RA, but not in AId or RA cup. Juvenile RA neurons were broadly responsive to multiple songs but preferred juvenile over adult vocal sounds; in addition, spiking responses lacked consistent temporal patterning. By adulthood, RA neurons responded most strongly to each bird's own song with precisely timed spiking activity. In contrast, we observed a complete lack of song responsivity in both juvenile and adult AId, even though this region receives song-responsive inputs. A surprisingly large proportion of sites in RA cup of both juveniles and adults did not respond to song playback, and responsive sites showed little evidence of song selectivity. NEW & NOTEWORTHY Motor skill learning entails changes in selectivity for behaviorally relevant stimuli across cortical regions, yet the neural representation of these stimuli remains understudied. We investigated how information important for vocal learning in zebra finches is represented in regions analogous to infragranular layers of motor and auditory cortices during vs. after the developmentally regulated learning period. The results provide insight into how neurons in higher level stages of cortical processing represent stimuli important for motor skill learning.

Response properties of single neurons in higher level auditory cortex of adult songbirds.

The caudomedial nidopallium (NCM) is a higher level region of auditory cortex in songbirds that has been implicated in encoding learned vocalizations and mediating perception of complex sounds. We made cell-attached recordings in awake adult male zebra finches ( Taeniopygia guttata) to characterize responses of single NCM neurons to playback of tones and songs. Neurons fell into two broad classes: narrow fast-spiking cells and broad sparsely firing cells. Virtually all narrow-spiking cells responded to playback of pure tones, compared with approximately half of broad-spiking cells. In addition, narrow-spiking cells tended to have lower thresholds and faster, less variable spike onset latencies than did broad-spiking cells, as well as higher firing rates. Tonal responses of narrow-spiking cells also showed broader ranges for both frequency and amplitude compared with broad-spiking neurons and were more apt to have V-shaped tuning curves compared with broad-spiking neurons, which tended to have complex (discontinuous), columnar, or O-shaped frequency response areas. In response to playback of conspecific songs, narrow-spiking neurons showed high firing rates and low levels of selectivity whereas broad-spiking neurons responded sparsely and selectively. Broad-spiking neurons in which tones failed to evoke a response showed greater song selectivity compared with those with a clear tuning curve. These results are consistent with the idea that narrow-spiking neurons represent putative fast-spiking interneurons, which may provide a source of intrinsic inhibition that contributes to the more selective tuning in broad-spiking cells. NEW & NOTEWORTHY The response properties of neurons in higher level regions of auditory cortex in songbirds are of fundamental interest because processing in such regions is essential for vocal learning and plasticity and for auditory perception of complex sounds. Within a region of secondary auditory cortex, neurons with narrow spikes exhibited high firing rates to playback of both tones and multiple conspecific songs, whereas broad-spiking neurons responded sparsely and selectively to both tones and songs.

Crosstalk between Estrogen Withdrawal and NFκB Signaling following Penetrating Brain Injury

Objectives: Characterized by neuroinflammation, traumatic brain injury (TBI) induces neuropathological changes and cognitive deficits. Estrogens are neuroprotective by increasing cell survival and this increase is mediated by a decrease in neuroinflammation. To further explore the relationship between estrogens, brain injury, and neuroinflammation, we examined the expression of the IKK/NFκB complex. The IKK/NFκB complex is a pleiotropic regulator of many cellular signaling pathways linked to inflammation, as well as three major cytokines (IL-1β, IL-6, and TNF-α). We hypothesized that NFκB expression would be upregulated following injury and that this increase would be exacerbated when circulating estrogens were decreased with fadrozole (aromatase inhibitor). Methods: Using adult zebra finches, we first determined the expression of major components of the NFκB complex (NFκB, IκB-α, and IκB-β) following injury using qPCR. Next, male and female finches were collected at 2 time points (2 or 24 h after injury) and brain tissue was analyzed to determine whether NFκB expression was differentially expressed in males and females at either time point. Finally, we examined how the expression of NFκB changed when estrogen levels were decreased immediately after injury. Results: Our study documented an increase in the expression of the major components of the NFκB complex (NFκB, IκB-α, and IκB-β) following injury. Decreasing estrogen levels resulted in a surprising decrease in the NFκB complex studied here. Discussion: These data further expand the model of how estrogens and other steroid hormones interact with the inflammatory pathways following injury and may prove beneficial when developing therapies for treatment of TBI.

The Avian Basal Ganglia Are a Source of Rapid Behavioral Variation That Enables Vocal Motor Exploration.

The basal ganglia (BG) participate in aspects of reinforcement learning that require evaluation and selection of motor programs associated with improved performance. However, whether the BG additionally contribute to behavioral variation ("motor exploration") that forms the substrate for such learning remains unclear. In songbirds, a tractable system for studying BG-dependent skill learning, a role for the BG in generating exploratory variability, has been challenged by the finding that lesions of Area X, the song-specific component of the BG, have no lasting effects on several forms of vocal variability that have been studied. Here we demonstrate that lesions of Area X in adult male zebra finches (Taeniopygia gutatta) permanently eliminate rapid within-syllable variation in fundamental frequency (FF), which can act as motor exploration to enable reinforcement-driven song learning. In addition, we found that this within-syllable variation is elevated in juveniles and in adults singing alone, conditions that have been linked to enhanced song plasticity and elevated neural variability in Area X. Consistent with a model that variability is relayed from Area X, via its cortical target, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), to influence song motor circuitry, we found that lesions of LMAN also eliminate within-syllable variability. Moreover, we found that electrical perturbation of LMAN can drive fluctuations in FF that mimic naturally occurring within-syllable variability. Together, these results demonstrate that the BG are a central source of rapid behavioral variation that can serve as motor exploration for vocal learning.SIGNIFICANCE STATEMENT Many complex motor skills, such as speech, are not innately programmed but are learned gradually through trial and error. Learning involves generating exploratory variability in action ("motor exploration") and evaluating subsequent performance to acquire motor programs that lead to improved performance. Although it is well established that the basal ganglia (BG) process signals relating to action evaluation and selection, whether and how the BG promote exploratory motor variability remain unclear. We investigated this question in songbirds, which learn to produce complex vocalizations through trial and error. In contrast with previous studies that did not find effects of BG lesions on vocal motor variability, we demonstrate that the BG are an essential source of rapid behavioral variation linked to vocal learning.

The effect of social context on measures of boldness: Zebra finches (Taeniopygia guttata) are bolder when housed individually

Boldness can be quantified as latency to access resources in novel contexts. Although social interactions influence boldness, especially in species such as zebra finches (Taeniopygia guttata) that are sociable and live in groups, the boldness of individuals is generally characterized in isolation. The objective of this study was to examine how proximity of and familiarity with flock members influenced boldness, which is often assumed to be stable across contexts. We measured boldness as the latency of adult zebra finches to forage when a novel object was within a food dish in five social contexts: (1) social isolation, (2) adjacent to novel flock, (3) adjacent to familiar flock, (4) within novel flock, (5) within familiar flock. Our measure of boldness showed individual consistency across social contexts. However, when mean response values across different social contexts were compared, individuals were bolder (i.e. had lower latencies) when housed and tested individually, regardless of the presence or absence of a flock nearby. We found no evidence of sex, dominance rank, or flock members’ boldness on the boldness of a focal bird. Our study deepens understanding of the influence of the social environment on boldness in zebra finches by demonstrating an influence of physical proximity. We encourage continued work examining boldness in different social contexts in zebra finches, as rigorous work in one system may help us understand the factors that influence the relative stability or flexibility of this trait.

Zebra finches are sensitive to combinations of temporally distributed features in a model of word recognition.

Discrimination between spoken words composed of overlapping elements, such as "captain" and "captive," relies on sensitivity to unique combinations of prefix and suffix elements that span a "uniqueness point" where the word candidates diverge. To model such combinatorial processing, adult female zebra finches were trained to discriminate between target and distractor syllable sequences that shared overlapping "contextual" prefixes and differed only in their "informative" suffixes. The transition from contextual to informative syllables thus created a uniqueness point analogous to that present between overlapping word candidates, where targets and distractors diverged. It was found that target recognition depended not only on informative syllables, but also on contextual syllables that were shared with distractors. Moreover, the influence of each syllable depended on proximity to the uniqueness point. Birds were then trained birds with targets and distractors that shared both prefix and suffix sequences and could only be discriminated by recognizing unique combinations of those sequences. Birds learned to robustly discriminate target and distractor combinations and maintained significant discrimination when the local transitions from prefix to suffix were disrupted. These findings indicate that birds, like humans, combine information across temporally distributed features, spanning contextual and informative elements, in recognizing and discriminating word-like stimuli.

Questioning Seasonality of Neuronal Plasticity in the Adult Avian Brain

To date, studies that reported seasonal patterns of adult neurogenesis and neuronal recruitment have correlated them to seasonal behaviors as the cause or as a consequence of neuronal changes. The aim of our study was to test this correlation, and to investigate whether there is a seasonal pattern of new neuronal recruitment that is not correlated to behavior. To do this, we used adult female zebra finches (songbirds that are not seasonal breeders), kept them under constant social, behavioral, and spatial environments, and compared neuronal recruitment in their brains during two seasons, under natural and laboratory conditions. Under natural conditions, no significant differences were found in the pattern of new neuronal recruitment across seasons. However, under artificial indoor conditions that imitated the natural conditions, higher neuronal recruitment occurred in late summer (August) compared to early spring (February). Moreover, our data indicate that “mixing” temperature and day length significantly reduces new neuronal recruitment, demonstrating the importance of the natural combination of temperature and day length. Taken together, our findings show, for the first time, that neuroplasticity changes under natural vs. artificial conditions, and demonstrate the importance of both laboratory and field experiments when looking at complex biological systems.

Pre-Bout Neural Activity Changes in Premotor Nucleus HVC Correlate with Successful Initiation of Learned Song Sequence.

Preparatory activity, characterized by gradual, longer timescale changes in neural activity, is present in a number of different brain areas before the onset of simple movements and is believed to be important for movement initiation. However, relatively little is known about such activity before initiation of naturally learned movement sequences. The song of an adult male zebra finch is a well studied example of a naturally learned movement sequence and previous studies have shown robust premotor activity immediately before song. Here, I characterize longer timescale changes in neural activity in adult male zebra finch premotor nucleus HVC before onset of song bouts. I show that interneurons and a subset of basal-ganglia-projecting neurons change their activity several hundred milliseconds before song bout onset. Interneurons increased their activity, whereas basal-ganglia-projecting neurons either increased or decreased their activity. Such changes in neural activity were larger, started earlier, and were more common specifically before song bouts that began with the short, repetitive, introductory notes (INs) characteristic of zebra finch song bouts. Further, stronger and earlier changes were also correlated with successful song sequence initiation. Finally, a small fraction of basal-ganglia-projecting neurons that increased their activity before song bout onset did not have song or IN-related activity, suggesting a specialized preparatory role for such neurons. Overall, these data suggest that pre-bout activity in HVC represents preparatory activity important for initiation of a naturally learned movement sequence.SIGNIFICANCE STATEMENT Changes in neuronal activity well before the onset of simple movements are thought to be important for movement initiation. However, a number of animal movements consist of sequences of simple movements and relatively little is known about neuronal activity before such movement sequences. Using adult zebra finch song, a well studied example of a movement sequence, I show here that neurons in premotor nucleus HVC change their activity hundreds of milliseconds before song bout onset. In most neurons, the presence of such changes correlated with successful song sequence initiation. My results show the presence of preparatory neural activity in HVC and suggest a role for HVC in sequence initiation in addition to its established role in song sequence timing.

Stable Sequential Activity Underlying the Maintenance of a Precisely Executed Skilled Behavior

A vast array of motor skills can be maintained throughout life. Do these behaviors require stability of individual neuron tuning or can the output of a given circuit remain constant despite fluctuations in single cells? This question is difficult to address due to the variability inherent in most motor actions studied in the laboratory. A notable exception, however, is the courtship song of the adult zebra finch, which is a learned, highly precise motor act mediated by orderly dynamics within premotor neurons of the forebrain. By longitudinally tracking the activity of excitatory projection neurons during singing using two-photon calcium imaging, we find that both the number and the precise timing of song-related spiking events remain nearly identical over the span of several weeks to months. These findings demonstrate that learned, complex behaviors can be stabilized by maintaining precise and invariant tuning at the level of single neurons.

Relative salience of syllable structure and syllable order in zebra finch song.

There is a rich history of behavioral and neurobiological research focused on the 'syntax' of birdsong as a model for human language and complex auditory perception. Zebra finches are one of the most widely studied songbird species in this area of investigation. As they produce song syllables in a fixed sequence, it is reasonable to assume that adult zebra finches are also sensitive to the order of syllables within their song; however, results from electrophysiological and behavioral studies provide somewhat mixed evidence on exactly how sensitive zebra finches are to syllable order as compared, say, to syllable structure. Here, we investigate how well adult zebra finches can discriminate changes in syllable order relative to changes in syllable structure in their natural song motifs. In addition, we identify a possible role for experience in enhancing sensitivity to syllable order. We found that both male and female adult zebra finches are surprisingly poor at discriminating changes to the order of syllables within their species-specific song motifs, butare extraordinarily good at discriminating changes to syllable structure (i.e., reversals) in specific syllables. Direct experience or familiarity with a song, either using the bird's own song (BOS) or the song of a flock mate as the test stimulus, improved both male and female zebra finches' sensitivity to syllable order. However, even with experience, birds remained much more sensitive to structural changes in syllables. These results help to clarify some of the ambiguities from the literature on the discriminability of changes in syllable order in zebra finches, provide potential insight on the ethological significance of zebra finch song features, and suggest new avenues of investigation in using zebra finches as animal models for sequential sound processing.

A complex mTOR response in habituation paradigms for a social signal in adult songbirds.

Nonassociative learning is considered simple because it depends on presentation of a single stimulus, but it likely reflects complex molecular signaling. To advance understanding of the molecular mechanisms of one form of nonassociative learning, habituation, for ethologically relevant signals we examined song recognition learning in adult zebra finches. These colonial songbirds learn the unique song of individuals, which helps establish and maintain mate and other social bonds, and informs appropriate behavioral interactions with specific birds. We leveraged prior work demonstrating behavioral habituation for individual songs, and extended the molecular framework correlated with this behavior by investigating the mechanistic Target of Rapamycin (mTOR) signaling cascade. We hypothesized that mTOR may contribute to habituation because it integrates a variety of upstream signals and enhances associative learning, and it crosstalks with another cascade previously associated with habituation, ERK/ZENK. To begin probing for a possible role for mTOR in song recognition learning, we used a combination of song playback paradigms and bidirectional dysregulation of mTORC1 activation. We found that mTOR demonstrates the molecular signatures of a habituation mechanism, and that its manipulation reveals the complexity of processes that may be invoked during nonassociative learning. These results thus expand the molecular targets for habituation studies and raise new questions about neural processing of complex natural signals.

Morphological characterization of HVC projection neurons in the zebra finch (Taeniopygia guttata).

Singing behavior in the adult male zebra finch is dependent upon the activity of a cortical region known as HVC (proper name). The vast majority of HVC projection neurons send primary axons to either the downstream premotor nucleus RA (robust nucleus of the arcopallium, or primary motor cortex) or Area X (basal ganglia), which play important roles in song production or song learning, respectively. In addition to these long-range outputs, HVC neurons also send local axon collaterals throughout that nucleus. Despite their implications for a range of circuit models, these local processes have never been completely reconstructed. Here, we use in vivo single-neuron Neurobiotin fills to examine 40 projection neurons across 31 birds with somatic positions distributed across HVC. We show that HVC(RA) and HVC(X) neurons have categorically distinct dendritic fields. Additionally, these cell classes send axon collaterals that are either restricted to a small portion of HVC ("local neurons") or broadly distributed throughout the entire nucleus ("broadcast neurons"). Overall, these processes within HVC offer a structural basis for significant local processing underlying behaviorally relevant population activity.