Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control.

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navß3 to Navß4. Dynamic clamping and dialysis of Navß4's C-terminal peptide into juvenile RA neurons provide evidence that Navß4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Intrinsic neuronal properties represent song and error in zebra finch vocal learning.

Neurons regulate their intrinsic physiological properties, which could influence network properties and contribute to behavioral plasticity. Recording from adult zebra finch brain slices we show that within each bird basal ganglia Area X-projecting (HVCX) neurons share similar spike waveform morphology and timing of spike trains, with modeling indicating similar magnitudes of five principal ion currents. These properties vary among birds in lawful relation to acoustic similarity of the birds' songs, with adult sibling pairs (same songs) sharing similar waveforms and spiking characteristics. The properties are maintained dynamically: HVCX within juveniles learning to sing show variable properties, whereas the uniformity rapidly degrades within hours in adults singing while exposed to abnormal (delayed) auditory feedback. Thus, within individual birds the population of current magnitudes covary over the arc of development, while rapidly responding to changes in feedback (in adults). This identifies network interactions with intrinsic properties that affect information storage and processing of learned vocalizations.

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.

Neuronal Intrinsic Physiology Changes During Development of a Learned Behavior.

Juvenile male zebra finches learn their songs over distinct auditory and sensorimotor stages, the former requiring exposure to an adult tutor song pattern. The cortical premotor nucleus HVC (acronym is name) plays a necessary role in both learning stages, as well as the production of adult song. Consistent with neural network models where synaptic plasticity mediates developmental forms of learning, exposure to tutor song drives changes in the turnover, density, and morphology of HVC synapses during vocal development. A network's output, however, is also influenced by the intrinsic properties (e.g., ion channels) of the component neurons, which could change over development. Here, we use patch clamp recordings to show cell-type-specific changes in the intrinsic physiology of HVC projection neurons as a function of vocal development. Developmental changes in HVC neurons that project to the basal ganglia include an increased voltage sag response to hyperpolarizing currents and an increased rebound depolarization following hyperpolarization. Developmental changes in HVC neurons that project to vocal-motor cortex include a decreased resting membrane potential and an increased spike amplitude. HVC interneurons, however, show a relatively stable range of intrinsic features across vocal development. We used mathematical models to deduce possible changes in ionic currents that underlie the physiological changes and to show that the magnitude of the observed changes could alter HVC circuit function. The results demonstrate developmental plasticity in the intrinsic physiology of HVC projection neurons and suggest that intrinsic plasticity may have a role in the process of song learning.

Testosterone Mediates Seasonal Growth of the Song Control Nuclei in a Tropical Bird.

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Sexual differences in cell proliferation in the ventricular zone, cell migration and differentiation in the HVC of juvenile Bengalese finch.

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.

Afferents from vocal motor and respiratory effectors are recruited during vocal production in juvenile songbirds.

Learned behaviors require coordination of diverse sensory inputs with motivational and motor systems. Although mechanisms underlying vocal learning in songbirds have focused primarily on auditory inputs, it is likely that sensory inputs from vocal effectors also provide essential feedback. We investigated the role of somatosensory and respiratory inputs from vocal effectors of juvenile zebra finches (Taeniopygia guttata) during the stage of sensorimotor integration when they are learning to imitate a previously memorized tutor song. We report that song production induced expression of the immediate early gene product Fos in trigeminal regions that receive hypoglossal afferents from the tongue and syrinx (the main vocal organ). Furthermore, unilateral lesion of hypoglossal afferents greatly diminished singing-induced Fos expression on the side ipsilateral to the lesion, but not on the intact control side. In addition, unilateral lesion of the vagus reduced Fos expression in the ipsilateral nucleus of the solitary tract in singing birds. Lesion of the hypoglossal nerve to the syrinx greatly disrupted vocal behavior, whereas lesion of the hypoglossal nerve to the tongue exerted no obvious disruption and lesions of the vagus caused some alterations to song behavior. These results provide the first functional evidence that somatosensory and respiratory feedback from peripheral effectors is activated during vocal production and conveyed to brainstem regions. Such feedback is likely to play an important role in vocal learning during sensorimotor integration in juvenile birds and in maintaining stereotyped vocal behavior in adults.

Recurrent interactions between the input and output of a songbird cortico-basal ganglia pathway are implicated in vocal sequence variability.

Complex brain functions, such as the capacity to learn and modulate vocal sequences, depend on activity propagation in highly distributed neural networks. To explore the synaptic basis of activity propagation in such networks, we made dual in vivo intracellular recordings in anesthetized zebra finches from the input (nucleus HVC, used here as a proper name) and output [lateral magnocellular nucleus of the anterior nidopallium (LMAN)] neurons of a songbird cortico-basal ganglia (BG) pathway necessary to the learning and modulation of vocal motor sequences. These recordings reveal evidence of bidirectional interactions, rather than only feedforward propagation of activity from HVC to LMAN, as had been previously supposed. A combination of dual and triple recording configurations and pharmacological manipulations was used to map out circuitry by which activity propagates from LMAN to HVC. These experiments indicate that activity travels to HVC through at least two independent ipsilateral pathways, one of which involves fast signaling through a midbrain dopaminergic cell group, reminiscent of recurrent mesocortical loops described in mammals. We then used in vivo pharmacological manipulations to establish that augmented LMAN activity is sufficient to restore high levels of sequence variability in adult birds, suggesting that recurrent interactions through highly distributed forebrain-midbrain pathways can modulate learned vocal sequences.

Morphology of axonal projections from the high vocal center to vocal motor cortex in songbirds.

Only birds that learn complex vocalizations have telencephalic brain regions that control vocal learning and production, including HVC (high vocal center), a cortical nucleus that encodes vocal motor output in adult songbirds. HVC projects to RA (robust nucleus of the arcopallium), a nucleus in motor cortex that in turn projects topographically onto hindbrain neurons innervating vocal muscles. Individual neurons projecting from HVC to RA (HVC(RA) ) fire sparsely to drive RA activity during song production. To advance understanding of how individual HVC neurons encode production of learned vocalizations, we reconstructed single HVC axons innervating RA in adult male zebra finches. Individual HVC(RA) axons were not topographically organized within RA: 1) axon arbors of HVC cell bodies located near each other sent branches to different subregions of RA, and 2) branches of single HVC axons terminated in different locations within RA. HVC(RA) axons also had a simple, sparse morphology, suggesting that a single HVC neuron activates a limited population of postsynaptic RA neurons. These morphological data are consistent with previous work showing that single HVC(RA) neurons burst sparsely for a brief period of time during the production of a song, indicating that ensembles of HVC(RA) neurons fire simultaneously to drive small temporal segments of song behavior. We also examined the morphology of axons projecting from HVC to RA cup, a region surrounding RA that receives input from auditory cortex. Axons projecting to RA cup also sent some branches into RA, suggesting direct integration between the sensory and motor circuits for song control.

The zebra finch paradox: song is little changed, but number of neurons doubles.

New neurons are added to the high vocal center (HVC) of adult males in seasonally breeding songbirds such as the canary (Serinus canaria) that learns new songs in adulthood, and the song sparrow (Melospiza melodia) that does not. In both cases, the new neurons numerically replace others that have died, resulting in a seasonal fluctuation in HVC volume and neuron number. Peaks in neuronal replacement in both species occur in the fall when breeding is over and song is variable. New neurons are added, too, to the HVC of zebra finches (Taeniopygia guttata) that do not learn new songs in adulthood and whose song remains stereotyped throughout the year. Here, we show that, in contrast to the observations in seasonal songbirds, neurons added to the zebra finch HVC are not part of a replacement process. Rather, they lead to a doubling in the number of neurons that project from HVC to the robust nucleus of the arcopallium (RA). As this happens, HVC volume remains constant and the packing density of its neurons increases. The HVC-RA neurons are part of a descending pathway that carries the pattern of learned song; some HVC-RA neurons are also responsive to song playback. The addition of HVC-RA neurons happens in zebra finches housed singly, but becomes more acute if the birds are housed communally. We speculate that new neurons added to the adult HVC may help with the production or perception of learned song, or both.

Identification of single neurons in a forebrain network.

Behaviors are generated from complex interactions among networks of neurons. Single-unit ensemble recording has been used to identify multiple neurons in functioning networks. These recordings have provided insight into interactions among neurons in local and distributed circuits. Recorded units in these ensembles have been classed based on waveform type, firing pattern, and physical location. To identify individual projection neurons in a cortical network, we have paired tetrode recording with antidromic stimulation. We developed techniques that enable antidromic identification of single units and study of functional interactions between these neurons and other circuit elements. These methods have been developed in the zebra finch and should be applicable, with potential modifications that we discuss here, to any neural circuit with defined subpopulations based on projection target. This methodology will enable elucidation of the functional roles of single identified neurons in complex vertebrate circuits.

Developmental changes in the sexually dimorphic expression of secretory carrier membrane protein 1 and its co-localisation with androgen receptor protein in the zebra finch song system.

The song system of zebra finches differs dramatically between the sexes in terms of both structure and function. Only males sing and the brain regions regulating the learning and production of this behaviour are far more developed in males than females. Mechanisms regulating sexual differentiation likely include both direct genetic and hormonal processes. Expression of both mRNA and the protein product for secretory carrier membrane protein 1 (SCAMP1), a sex chromosome gene, are increased in the brains of juvenile males compared to females. Here we investigated developmental changes in SCAMP1 containing cells in song nuclei and co-localisation with androgen receptor (AR) protein from post-hatching day 25 through adulthood. Almost all SCAMP1 cells co-expressed AR and approximately half of the AR cells expressed SCAMP1 in the HVC and robust nucleus in the arcopallium (RA) of both sexes and in the Area X of males (which could not be clearly defined in females). In HVC and RA, more single and double-labelled cells were detected in males than females overall, and the sex differences increased as animals matured. The results suggest the potential for interaction of these two proteins in regulating development of brain and/or behaviour.

Neuroprotective effects of testosterone in a naturally occurring model of neurodegeneration in the adult avian song control system.

Seasonal regression of the avian song control system, a series of discrete brain nuclei that regulate song learning and production, serves as a useful model for investigating the neuroprotective effects of steroids. In seasonally breeding male songbirds, the song control system regresses rapidly when males are transferred from breeding to nonbreeding physiological conditions. One nucleus in particular, the HVC, regresses in volume by 22% within days of castration and transfer to a nonbreeding photoperiod. This regression is mediated primarily by a 30% decrease in neuron number, a result of a caspase-dependent process of programmed cell death. Here we examine whether testosterone (T) can act locally in the brain to prevent seasonal-like neurodegeneration in HVC. We began to infuse T intracerebrally near HVC on one side of the brain in breeding-condition male white-crowned sparrows 2 days prior to T withdrawal and shifting them to short-day photoperiods. The birds were killed 3 or 7 days later. Local T infusion significantly protected ipsilateral HVC from volume regression and neuron loss. In addition, T infusion significantly reduced the number, density, and number/1,000 neurons of activated caspase-3 cells and cells positive for cleaved PARP, both markers for programmed cell death, in the ipsilateral HVC. T infusion near HVC also prevented regression of ipsilateral efferent targets of HVC neurons, including the volumes of robust nucleus of the arcopallium (RA) and Area X and the soma area and density of RA neurons. Thus T can act locally in the brain to have a neuroprotective effect and act transsynaptically to prevent regression of efferent nuclei.

Sexual dimorphism of the electrophysiological properties of the projection neurons in the robust nucleus of the arcopallium in adult zebra finches.

OBJECTIVE: To observe the sexual differences in electrophysiological properties of neurons in the robust nucleus of the arcopallium (RA) in adult zebra finches, and to provide the direct electrophysiological evidence for the sexual dimorphism of birdsong. METHODS: Whole-cell recording was used to record the spontaneous action potential firing rates from RA projection neurons in acute brain slices. RESULTS: The projection neurons of RA in male birds fired spontaneously at 10 Hz or above, while in female birds, the frequency was significantly lower, and even no firings could be detected. CONCLUSION: There is a sexual difference in electrophysiological properties of projection neurons in RA, which may result from the difference in the levels of steroid hormones in birds.

Neuronal stability and drift across periods of sleep: premotor activity patterns in a vocal control nucleus of adult zebra finches.

How stable are neural activity patterns compared across periods of sleep? We evaluated this question in adult zebra finches, whose premotor neurons in the nucleus robustus arcopallialis (RA) exhibit sequences of bursts during daytime singing that are characterized by precise timing relative to song syllables. Each burst has a highly regulated pattern of spikes. We assessed these spike patterns in singing that occurred before and after periods of sleep. For about half of the neurons, one or more premotor bursts had changed after sleep, an average of 20% of all bursts across all RA neurons. After sleep, modified bursts were characterized by a discrete, albeit modest, loss of spikes with compensatory increases in spike intervals, but not changes in timing relative to the syllable. Changes in burst structure followed both interrupted bouts of sleep (1.5-3 h) and full nights of sleep, implicating sleep and not circadian cycle as mediating these effects. Changes in burst structure were also observed during the day, but far less frequently. In cases where multiple bursts in the sequence changed in a single cell, the sequence position of those bursts tended to cluster together. Bursts that did not show discrete changes in structure also showed changes in spike counts, but not biased toward losses. We hypothesize that changes in burst patterns during sleep represent active sculpting of the RA network, supporting auditory feedback-mediated song maintenance.

Inhibition and recurrent excitation in a computational model of sparse bursting in song nucleus HVC.

The telencephalic premotor nucleus HVC is situated at a critical point in the pattern-generating premotor circuitry of oscine songbirds. A striking feature of HVC's premotor activity is that its projection neurons burst extremely sparsely. Here we present a computational model of HVC embodying several central hypotheses: 1) sparse bursting is generated in bistable groups of recurrently connected robust nucleus of the arcopallium (RA)-projecting (HVCRA) neurons; 2) inhibitory interneurons terminate bursts in the HVCRA groups; and 3) sparse sequences of bursts are generated by the propagation of waves of bursting activity along networks of HVCRA neurons. Our model of sparse bursting places HVC in the context of central pattern generators and cortical networks using inhibition, recurrent excitation, and bistability. Importantly, the unintuitive result that inhibitory interneurons can precisely terminate the bursts of HVCRA groups while showing relatively sustained activity throughout the song is made possible by a specific constraint on their connectivity. We use the model to make novel predictions that can be tested experimentally.

Wireless neural stimulation in freely behaving small animals.

We introduce a novel wireless, low-power neural stimulation system for use in freely behaving animals. The system consists of an external transmitter and a miniature, implantable wireless receiver-stimulator. The implant uses a custom integrated chip to deliver biphasic current pulses to four addressable bipolar electrodes at 32 selectable current levels (10 microA to 1 mA). To achieve maximal battery life, the chip enters a sleep mode when not needed and can be awakened remotely when required. To test our device, we implanted bipolar stimulating electrodes into the songbird motor nucleus HVC (formerly called the high vocal center) of zebra finches. Single-neuron recordings revealed that wireless stimulation of HVC led to a strong increase of spiking activity in its downstream target, the robust nucleus of the arcopallium. When we used this device to deliver biphasic pulses of current randomly during singing, singing activity was prematurely terminated in all birds tested. Thus our device is highly effective for remotely modulating a neural circuit and its corresponding behavior in an untethered, freely behaving animal.

Neural correlates of categorical perception in learned vocal communication.

The division of continuously variable acoustic signals into discrete perceptual categories is a fundamental feature of vocal communication, including human speech. Despite the importance of categorical perception to learned vocal communication, the neural correlates underlying this phenomenon await identification. We found that individual sensorimotor neurons in freely behaving swamp sparrows expressed categorical auditory responses to changes in note duration, a learned feature of their songs, and that the neural response boundary accurately predicted the categorical perceptual boundary measured in field studies of the same sparrow population. Furthermore, swamp sparrow populations that learned different song dialects showed different categorical perceptual boundaries that were consistent with the boundary being learned. Our results extend the analysis of the neural basis of perceptual categorization into the realm of vocal communication and advance the learned vocalizations of songbirds as a model for investigating how experience shapes categorical perception and the activity of categorically responsive neurons.

Top-down regulation of plasticity in the birdsong system: "premotor" activity in the nucleus HVC predicts song variability better than it predicts song features.

We studied real-time changes in brain activity during active vocal learning in the zebra finch songbird. The song nucleus HVC is required for the production of learned song. To quantify the relationship of HVC activity and behavior, HVC population activity during repeated vocal sequences (motifs) was recorded and temporally aligned relative to the motif, millisecond by millisecond. Somewhat surprisingly, HVC activity did not reliably predict any vocal feature except amplitude and, to a lesser extent, entropy and pitch goodness (sound periodicity). Variance in "premotor" HVC activity did not reliably predict variance in behavior. In contrast, HVC activity inversely predicted the variance of amplitude, entropy, frequency, pitch, and FM. We reasoned that, if HVC was involved in song learning, the relationship of HVC activity to learned features would be developmentally regulated. To test this hypothesis, we compared the HVC song feature relationships in adults and juveniles in the sensorimotor "babbling" period. We found that the relationship of HVC activity to variance in FM was developmentally regulated, with the greatest difference at an HVC vocalization lag of 50 ms. Collectively, these data show that, millisecond by millisecond, bursts in HVC activity predict song stability on-line during singing, whereas decrements in HVC activity predict plasticity. These relationships between neural activity and plasticity may play a role in vocal learning in songbirds by enabling the selective stabilization of parts of the song that match a learned tutor model.

Population coding of song element sequence in the Bengalese finch HVC.

Birdsong is a complex vocalization composed of various song elements organized according to sequential rules. Two alternative views exist that explain the neural representation of song element sequences in the songbird brain. The finding of sequential selective neurons supports the idea that the song element sequence is encoded in a chain of rigid selective neurons. Alternatively, song structure could be encoded in an ensemble of relatively broad selective neurons arranged in a distributed manner. Here we attempted to determine which neural representation actually occurs in the song system by recording neural responses to various stimuli and performing information-theoretic analysis on the data obtained. We recorded the neural responses to all possible element pairs of stimuli in the Bengalese finch brain nucleus high vocal centre (HVC). Our results showed that each neuron has broad but differential response properties to element sequences beyond the structure of self-generated song. To quantify the transmitted information by such a broadly tuned neural population, we calculated the time course of mutual information between auditory stimuli and neural activities. Confounded information, which represents the relationship between present and previous elements, increased significantly immediately after stimulus presentation. These results indicate that the song element sequence is encoded in a neural ensemble in the HVC via population coding. These findings give us a new encoding scheme for the song element sequence using a distributed neural representation rather than the chain model of rigid selective neurons.