Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches

Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches Linda Bistere, Stefan Wilczek, Daniela Vallentin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4789872/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2024 Read the published version in BMC Neuroscience → Version 1 posted 4 You are reading this latest preprint version Abstract Zebra finches undergo a gradual refinement of their vocalizations, transitioning from variable juvenile songs to the stereotyped song of adulthood. To investigate the neural mechanisms underlying song crystallization, a critical phase in this developmental process, we conducted intracellular recordings in HVC, a premotor nucleus essential for song learning and production. We found that HVC projection neurons in juvenile zebra finches in the song crystallization phase exhibited more variable spiking patterns compared to the precise bursting observed in adult HVC projection neurons. Additionally, subthreshold membrane potential fluctuations in juvenile neurons were characterized by longer duration and larger amplitude excitatory postsynaptic potentials. These distinct temporal dynamics in HVC during song crystallization likely play a crucial role in the fine-tuning processes that shape the precise timing and structure of the mature zebra finch song. vocal learning HVC songbirds Figures Figure 1 Figure 2 Figure 3 Introduction The process of acquiring a motor skill encompasses the refinement of a once highly variable movement into its finely-tuned and precise execution. For example, the initial stages of vocal learning in humans are often characterized by inherently variable vocalizations such as cries and babbling sounds 1 . After months of practicing these utterances shape into refined words with precise pronunciation 2 . Male zebra finches undergo a comparable learning process for singing 3 . Within a critical period, their initially variable subsong transitions into a plastic song, ultimately culminating in the acquisition of a final, stereotyped song closely resembling that of their tutor 4 . Using bioacoustics, the song learning progression of developing zebra finches can be described by assessing changes in spectral features or temporal characteristics of song 4 – 6 . Subsong (~ 25–50 days post hatch (dph)) consists of poorly structured sounds with high variability of spectral features (wiener entropy, spectral continuity, pitch and frequency modulation) 7 . Subsong is followed by a plastic song (~ 50–80 dph) consisting of structured syllable production that gradually develops distinct acoustic features until a stereotyped, crystalized song is achieved which is characterized by precise, reliably repeated features 7 . In adults the spectral and temporal features of song are highly stereotyped and acoustic parameters have low variability. Throughout the developmental phase, neural dynamics in the vocal production pathway undergo various modifications 8 – 13 , ultimately leading to the emergence of a stable neural pattern during song production 14 – 19 . Initially the lateral magnocellular nucleus of the anterior neostriatum (LMAN) is necessary for the production and timing of subsong vocal patterns 8 . LMAN projects to RA (robust nucleus of the arcopallium) which in turn projects to the brainstem vocal and respiratory nuclei 20 , leading to audible song production. During the song learning phase, the main input to RA switches from LMAN to HVC, which on a behavioral level is reflected by increased stereotypy in timing and spectral features of song 21 . It has been shown to be a gradual and overlapping transition since HVC inactivation during the plastic song phase reverts the juveniles singing into subsong, whereas LMAN inactivation leads to the production of adult like stereotyped song sequences 10 , 22 . We aimed to explore the neural underpinnings that might fine-tune temporal dynamics of plastic song to eventually become a stereotyped song. Between 70 and 90 days post hatch the temporal structure of the song changes i.e. the silent gap duration decreases and the overall timing variability is reduced 23 . While RA is necessary for song production throughout all stages of development, its basic spiking characteristics develop concomitantly with spectral features and it does not generate timing 24 . LMAN is inducing temporal variability, but only during the subsong phase, since after ~ 60 dph, the RA motor program is predominantly driven from HVC 8 . Although HVC is not necessary for subsong, it is necessary for all later stages of song production and it has been shown to generate the timing of song in adults 24 and juveniles, in which HVC lesions result in the abolishment of adult like gap and syllable durations and HVC cooling results in an elongation of syllables and gaps 13 . During the early plastic song phase, HVC projection neurons produce rhythmic bursts during several syllables 11 . As the song matures, the bursting activity becomes tied to one particular timepoint during song 11 , but how these neural changes relate to alterations in the temporal features of singing behavior remains unclear. Here we directly investigated the neural activity changes at a single cell level that underly the transition from plastic song to crystalized song. We quantified the neural dynamics including changes in spiking characteristics and the synaptic inputs generated by connecting neurons by analyzing subthreshold membrane potential in singing zebra finches. Ultimately the crystallized song is sung by adult males to attract females, who prefer a highly temporally precise song. Results HVC projection neurons in juveniles exhibit more variable spiking and bursting patterns compared to adults The song development of zebra finches involves a gradual refinement of vocalizations, transitioning from variable juvenile songs to a stable, stereotyped adult form. During the crystallization phase, juveniles begin to produce structured syllables, signaling progress towards their final song. However, the temporal features of their songs, such as the timing between syllables, remain immature and undergo further refinement in the sensorimotor phase 23 . To understand the neural mechanisms underlying this temporal refinement, we focused on HVC, a premotor nucleus crucial for song learning and production. In adult zebra finches, the HVC motor program for song consists of precise, time-locked bursts of activity 14 – 16 . We aimed to investigate how this program develops during the plastic song phase, a stage of late-stage song learning. To achieve this, we conducted intracellular recordings of HVC projection neurons in freely behaving male juvenile zebra finches (74–94 days post hatch) during singing. Given HVC's role in controlling syllable and gap duration during this phase 13 , 24 , we analyzed the neural activity of HVC projection neurons in both juveniles and adults to identify potential differences that could contribute to the ongoing refinement of song timing. We found that HVC projection neurons elicited sparse bursts of action potentials during a specific time point during song production (Fig. 1 A, 1 B). The distribution of bursts spanned the entire song motif in both age groups 15 , 16 (Fig. 1 A, 1 B). This observation suggests that the neural dynamics underlying song production are already established during the late phase of song learning, leading to the production of stereotyped song structure. However, this finding alone cannot account for the observed differences in temporal variability between juvenile and adult songs 23 , indicating that other factors contribute to the ongoing refinement of song timing. It has been previously shown that in adult zebra finches HVC projection neurons display a sparse number of bursts during song production 14 – 16 , 24 – 26 . To explore whether our recorded neurons produced ultra-sparse bursting patterns as well, we calculated the maximum number of bursts per trial for each HVC projection neuron. In juveniles we observed a larger number of bursts during motif per HVC projection neuron when compared to adults (maximum number of bursts per motif: juveniles = 0–4 bursts per motif, median = 2, 10 neurons in 4 juveniles; adults = 0–4 bursts per motif, median = 1, 54 neurons in 10 adults, p = 0.03, Wilcoxon rank sum test, Fig. 1 C). Next, we measured the degree of stereotypy of bursting activity across song motifs. We identified bursts as reoccurring bursts across song motifs, if their onset time was within ± 20ms across motifs. In adults, 85.94% of all recorded bursts were consistently repeated across multiple renditions of the song motif, indicating a high degree of stereotypy. Conversely, in juveniles, only 60% of bursts were reoccurring (number of reoccurring bursts, juveniles = 12/20 bursts, adults = 55/64 bursts, p = 0.02, Fisher Exact Test). These observations are in line with previous work 11 and suggest, that the functional connectivity within HVC exhibits greater variability in late-stage juveniles compared to adults. In addition to bursting activity some neurons also exhibited single action potentials during song production which reduces the sparseness of the neural code and might induce less reliable behavioral outcomes in terms of song consistency and timing. To test whether single action potentials might contribute to a less temporally stereotyped song we explored the spiking activity of the HVC neurons (spikes within a burst and single spikes). We observed, that the number of spikes per motif tended to be higher in juvenile birds than in adults despite not significantly so (spikes per motif: juveniles = 6.5 spikes/motif, adults = 4 spikes/motif, p = 0.08, one-way ANOVA, Fig. 1 D). Higher number of spikes per motif could also be attributed to single spikes alone outside of bursts. To account for the potential impact of single spikes, we separately analyzed their occurrence across song renditions. This analysis revealed that HVC neurons in juvenile zebra finches exhibited a significantly higher number of single action potentials compared to adults (single spikes per motif: juveniles = 0.25 single spikes/motif, adults = 0 single spikes/motif, p = 0.03, one-way ANOVA, Fig. 1 E). The number of spikes recruited for bursting activity per song motif did not differ between juveniles and adults on a single neuron level (number of spikes within bursts: juveniles = 5.5 ± 3.54, median = 5.25, adults = 3.91 ± 3.96, median = 3.75, p = 0.23, one-way ANOVA, Fig. 1 F). These results show that in late-stage development HVC projection neurons in juveniles exhibit detectable differences in spiking activity compared to adults. However, this increased spiking activity in juveniles is not reflected in the spiking patterns within bursts themselves. Instead, it can be attributed to a higher frequency of single spikes occurring outside of bursts. This increased incidence of single spikes might contribute to the greater variability observed in juvenile song production. We next explored, whether the song variability in juveniles during the late-state development can also be attributed to temporal dynamics within bursts in HVC projection neurons. Temporal dynamics of HVC neuron bursting activity are slower in juveniles compared to adults We explored the intrinsic dynamics of individual bursts by analyzing their individual number of spikes and temporal characteristics of these spikes during song production (Fig. 2 A, 2 B). First, we quantified the number of spikes occurring per burst. In both juveniles and adults, we observed a similar number of spikes occurring per burst (median spikes per burst ± std: juveniles = 3 ± 1.95, adults = 3 ± 1.94, p = 0.26, Wilcoxon rank sum test, Fig. 2 C). Next, we assessed the variability of the number of spikes occurring in each reoccurring burst. HVC projection neurons in juveniles had a comparable distribution of Δ number of spikes per burst to neurons in adults (Δ number of spikes per burst: juveniles = 0.32 ± 0.61, adults = 0.37 ± 0.29, p = 0.09, Wilcoxon rank sum test, Fig. 2 D), which indicates stereotyped reoccurring bursts during the late-stage development. We next investigated whether the temporal structure of bursts in juveniles and adults differed by quantifying their duration. Unlike previously reported 11 , we did not observe differences in the duration of bursts between juveniles and adults on a population level potentially due to our smaller sample size (duration of bursts: juveniles = 9.64 ms, adults = 6.83 ms, Wilcoxon rank sum test, p = 0.15, Fig. 2 E). However, when assessing the instantaneous firing rate within bursts, we found that it was lower in juveniles than adults (firing rate: juveniles = 226.09 Hz, adults = 387.68 Hz, p < 0.001, Wilcoxon rank sum test, Fig. 2 F) indicating that premotor signals are produced on a slower timescale compared to in the adult brain. To quantify whether the lower instantaneous firing rate also produced a distinct temporal pattern of spiking progression within bursts, we next compared the normalized pattern of spiking progression in juveniles and adults (Fig. 2 G). Once we accounted for the overall higher instantaneous firing rate in adults by demeaning the progression pattern in juveniles and adults, the relative progression in juveniles was not distinguishable from that of the adult HVC projection neurons (demeaned firing rate: juveniles = 3.41*10 − 14 ±25.45 Hz, adults=-1.14*10 − 14 ±55.04 Hz, p = 0.84, Wilcoxon rank sum test, Fig. 2 H). To verify, whether the synaptically connected neurons provide stereotyped excitatory inputs preceding the bursts, we analyzed the membrane potential rise during a 15 ms window preceding all recorded bursts. Excitatory input accounting to the membrane potential rise preceding the bursts was as stereotyped in singing juveniles as in quiet juveniles where we elicited bursts using current injection (Wilcoxon rank sum test, p = 0.21) or in singing adults (Wilcoxon rank sum test, p = 0.11, Supplementary Fig. 1). This finding indicates, that the elicitation of bursts in singing juveniles occurs in a stereotyped way that is comparable to adults. The overall stereotyped characteristics of bursts (i.e., number of spikes, burst duration and stereotyped excitatory input) and the lower instantaneous firing rate indicate precise yet slower signal transmission in juvenile HVC projection neurons than in adults. We hypothesized, that these temporal dynamics might also be exhibited in a focal microcircuit within HVC. Here we leveraged the ability to quantify subthreshold activity of individual HVC projection neurons. This metric reflects the integrated input received by a focal neuron from its presynaptic network. HVC projection neurons receive temporally distinct subthreshold inputs in juveniles compared to in adults To quantify the temporal dynamics of inputs that HVC projection neurons are receiving, we assessed the stereotypy of the membrane potential by correlating subthreshold activity across motif renditions for each recorded neuron (Fig. 3 A). In juveniles, the subthreshold activity was as stereotyped as in adults (subthreshold precision: juveniles = 0.82, adults = 0.83, p = 0.59, Wilcoxon rank sum test, Fig. 3 B), suggesting a stable neural representation of song production. Since we previously reported temporal differences in bursting activity, we hypothesized, that these differences could also be reflected in the summation of excitatory and inhibitory postsynaptic potentials (PSPs) received by a focal neuron. To address these temporal dynamics, we quantified the duration of the PSPs and observed, that PSP events in juveniles were of longer duration than in adults (PSP duration: juveniles = 21.11 ± 2.94, adults = 18.09 ± 3.54, p = 0.01, Wilcoxon rank sum test, Fig. 3 C), which is in line with our previously observed slower temporal dynamic within bursts in juveniles. Further we quantified the membrane potential amplitude, and found that PSPs in juveniles had a higher membrane potential amplitude than in adults (PSP amplitude: juveniles = 6.37 ± 1.2mV, adults = 5.64 ± 1.43, p < 0.01, Wilcoxon rank sum test, Fig. 3 D), which could potentially be accounted to a higher resting membrane potential in juveniles 27 . In juveniles, a higher resting membrane potential is associated with altered neuronal excitability and could likely manifest as changes in the frequency or amplitude of postsynaptic potential events. The frequency of the PSP element occurrence was lower in juveniles than adults (PSP element frequency: juveniles = 12.85 ± 4.99, adults = 16.03 ± 4.91, p = 0.045, Wilcoxon rank sum test, Fig. 3 E), suggesting a slower-paced, more sparse input from synaptically connected neurons. The slower and more sparse membrane potential dynamics during singing in juveniles might explain the temporal differences during song performance 4 , 23 . Discussion In this study, we leveraged the developmental transition in song production—from the variable plastic song of juvenile zebra finches to the crystallized song of adults—to investigate the neural mechanisms underlying this behavioral shift. We focused specifically on the HVC, a premotor nucleus critical for song learning and production, to understand the neural dynamics that facilitate this transformation. The song crystallization phase in late-stage juveniles is characterized by a higher number of bursts of HVC projection neurons during singing, that were more variable in their occurrence than bursts of HVC projection neurons in adult birds (Fig. 1 ). Such neural pattern may underlie the more variable and exploratory vocalizations observed in juveniles during song learning. Despite receiving stereotyped excitatory input, bursts in juveniles exhibited a lower instantaneous firing rate than in adults (Fig. 2 ). This finding suggests that developmental changes in intrinsic neuronal properties may play a role in how the temporal information is encoded within HVC. Further, the stereotyped post-synaptic events occurring at a lower frequency in juveniles (Fig. 3 ) may indicate slower dynamics in their excitatory drive. In adult birds, HVC-RA projection neurons burst sparsely during singing 14 . These bursts are triggered by presynaptic inputs with a range of latencies to form neural sequences during song production 19 . In juvenile birds, the connectivity in HVC develops over time by bursting events gradually becoming more timepoint-specific during singing 11 . In this study, we report that the bursting activity in the late-stage juveniles occurred more frequently with less stereotypy across motifs than in adults. The development of song in zebra finches is accompanied by a progressive increase in inhibition within the HVC 28 . In adult birds, the precise activity of inhibitory neurons is crucial for shaping the sparse bursts of excitatory neurons within HVC, which are thought to contribute to the stereotyped nature of adult song 29 . It is plausible that the excitatory-inhibitory balance within the HVC of the juvenile birds we recorded from was not yet fully matured, potentially contributing to the observed differences in bursting activity compared to adults. In terms of subthreshold activity, neural dynamics were stable during song production in both juveniles and adults, which is in line with a previous study reporting highly stereotyped song performance of female-directed songs in juveniles, that were more variable during song practice 30 . Additionally, the higher amplitude post-synaptic potentials in juveniles could be another indicator of lower inhibitory current within HVC during song performance. Alternatively, neural dynamics may also have distinct temporal signatures due to different membrane properties of the HVC projection neurons in juveniles and adults. During development, the membrane properties of HVC projection neurons exhibit a higher resting membrane potential and an increased spiking amplitude 27 , which could lead to a higher general excitability of the HVC projection neurons. In line with this hypothesis, we report an increased amplitude of the excitatory post-synaptic events in juveniles. These excitatory post-synaptic events occurred at a lower frequency during singing in juveniles compared to in adults. The slower temporal dynamics could be attributed to incomplete myelination of axonal connections and changing membrane properties during development 27 , that ultimately impact the signal propagation within HVC during the juvenile stage. In line with this hypothesis, studies in adults demonstrate relatively slow HVC projection neuron conduction velocities 19 . Additionally, intrinsic properties of a subset of HVC projection neurons exhibit higher variability during song learning than in adulthood 31 , likely leading to a higher variability in the neural dynamics within HVC during song production. Investigating the signal propagation within HVC during development would yield more insights as to whether it also plays a role into HVC network dynamics during song learning. We revealed distinct temporal dynamics within HVC during the song crystallization phase, where temporal and spectral features of the song are being fine-tuned. Despite the differences in temporal dynamics of HVC projection neurons during the song crystallization phase and adulthood, the overall stereotypy of neural patterns during song production remained stable. However, the observed differences in spiking and subthreshold activity between juvenile and adult HVC projection neurons underscore the dynamic nature of neural circuits during song learning. This suggests that HVC undergoes significant plasticity during development to establish the stable, stereotyped patterns associated with mature song production. Materials and Methods Animal housing All procedures were approved by the Regierungspräsidium Oberbayern (VET 02-21-201), Landesamt für Gesundheit und Soziales (LAGeSo Berlin) (G 0225/16) at the Freie Universität Berlin or according to Institutional Animal Care and Use Committee at New York University Langone Medical Center guidelines. Juvenile zebra finches (n = 4, older than 73 dph) were housed in an aviary or a breeding cage with their genetic parents up to 60 dph, at which they were moved to an adjacent aviary where they had visual and auditory contact with their parents. Adult male birds (n = 10, older than 100 dph) were acquired from a breeder. Surgery Male zebra finches were anesthetized with isoflurane (concentration: 1–3% isoflurane, 97–99% oxygen) at a 65° head angle. An incision was made to expose the skull and a square shaped area of trabecular bone was removed above HVC, RA and cerebellum using a dental drill (carbide bur, FG ¼, Johnson-Promident). Nucleus RA was targeted according to coordinates (0 point at the bifurcation of the midsaggital sinus, RA coordinates: posterior 1.85 mm, lateral 2.25 mm, ventral 1.8 mm). A carbon fiber electrode (Kation Scientific, LLC) was used to identify RA based on the firing pattern 18 , 32 . Next, we targeted HVC using coordinates (0 point at the bifurcation of the midsaggital sinus, HVC coordinates: anterior 0.2 mm, lateral 2.3 mm, ventral 0.2 mm) and confirmed the location with antidromic stimulation from RA 14 . A previously assembled microdrive for intracellular recordings 25 was then implanted at 25° angle above HVC for intracellular recordings. Birds were let to recover 1–3 days before the intracellular recordings. Intracellular recordings in freely behaving animals To record intracellularly we used sharp intracellular electrodes (borosilicate glass with filament, 0.1 mm diameter), that were previously pulled using a micropipette puller (Model P-97, Sutter Instrument). Before use, each electrode was backfilled with potassium acetate (concentration: 3M). Using a silicone elastomer, we built a well around the craniotomy in HVC and filled it with phosphate buffered saline (PBS). Next, we removed dura using a dura pick. While birds were in a head-fixed setting, we lowered down the glass electrode in HVC until we could identify surrounding cells using oscilloscope and an audio monitor. We then transferred the bird into the cage of the recording setup and gradually lowered the electrode within HVC, accompanied by a brief buying pulse (10–20 ms), until a successful penetration of a neuron. Next, we presented a female bird to the male bird to motivate singing behavior. For this study, we only selected neurons that had at least 30 mV action potentials, the recording lasted at least 3 minutes and the membrane potential was below − 50 mV. We could identify HVC projection neurons based on their low firing rate 25 and characteristic waveform. Song acquisition We briefly housed the juvenile and adult birds in sound attenuated boxes together with a female to motivate singing. Once song was recorded, we took one motif from each bird and detected syllables and gaps using threshold-based detection from the sound envelope. Durations of each segment were calculated by subtracting onset timing from the offset timing respectively. Spike and burst detection Spikes in each recorded trace were detected as events exceeding the 15 mV threshold from the demeaned trace. We defined bursts as single events where the firing rate of adjacent spikes exceeded 100 Hz and the spikes occurred on the same membrane depolarization event. To compare reoccurring bursts, we defined a burst as reoccurring, if the onset time of the burst (first spike) in next trial occurred ± 20 ms from the onset of burst in the previous trial. Delta of spikes per burst was calculated for each identified, reoccurring burst. First, the mode of spikes per burst was calculated for each reoccurring burst. Then, the number of spikes of each burst of the same identity was subtracted from mode and an absolute mean delta of number of spikes was calculated per identified burst. Instantaneous firing rate Instantaneous firing rate was calculated as the mean firing rate per reoccurring burst and was defined as number of inter spike interval (ISI) divided by the sum of ISIs per burst. To make a fair comparison of the temporal progression of spiking within bursts, we only considered bursts with no more than 6 spikes per burst for this analysis. Excitatory post-synaptic potential detection For detection of the PSPs, we first detected local peaks and events of membrane depolarization exceeding 2 mV amplitude, then measured their amplitude and calculated the frequency by dividing the number of events with the duration of the song motif. For the analysis of PSP duration, we only included PSPs that were shorter than 35 ms to avoid false PSP duration detection. Subthreshold stereotypy Subthreshold stereotypy was defined as a cross correlation at 0 lag of all trials within a cell. First, we cut the spikes of our recorded traces (spike detection as described above). We then demeaned all subthreshold traces for a fair comparison. Each trace was then correlated with every other trace from the same HVC projection neuron. Declarations Author Contributions: L.B. and D.V. conceived the study and designed the experiments. L.B. and S.W. conducted the experiments. L.B. analyzed the data. L.B. and D.V. wrote the manuscript, D.V. acquired funding and supervised the project. Competing Interest Statement: No competing interests. Ethical Approval and consent to participate: All procedures were approved by the Regierungspräsidium Oberbayern (VET 02-21-201), Landesamt für Gesundheit und Soziales (LAGeSo Berlin) (G 0225/16) at the Freie Universität Berlin or according to Institutional Animal Care and Use Committee at New York University Langone Medical Center guidelines. Consent to Publication: The authors hereby give our consent for the publication of the manuscript titled "Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches” in BMC Neuroscience We confirm that we have had the opportunity to review the manuscript and understand that it will be made publicly available upon publication. Funding: This work was supported by the HORIZON EUROPE European Research Council (ERC)-2017-StG-757459 MIDNIGHT, the Deutsche Forschungsgemeinschaft VA742/2-1 and the Deutsche Forschungsgemeinschaft 327654276–SFB 1315—awarded to D.V. 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Proceedings of the National Academy of Sciences 108 , 1687–1692 (2011). Daou, A. & Margoliash, D. Intrinsic neuronal properties represent song and error in zebra finch vocal learning. Nature Communications 11 , (2020). Spiro, J. E., Dalva, M. B. & Mooney, R. Long-range inhibition within the zebra finch song nucleus RA can coordinate the firing of multiple projection neurons. Journal of Neurophysiology 81 , 3007–3020 (1999). Additional Declarations No competing interests reported. Supplementary Files SupplFigure1BistereetalBMC.ai Supplementary Figure 1: Subthreshold activity preceding bursts. A) Example recording of an HVC projection neuron in a singing juvenile bird (78 days post hatch), aligned to burst onsets across four song motifs. Green lines indicate the selected interval of analysis 15 ms before burst onset. B) Subthreshold activity from the four traces (grey) of the example in A 15 ms window before burst onset. In orange – average subthreshold activity of the 15 ms interval. C) Mean subthreshold stereotypy of all pre-burst intervals per recorded neuron (quiet juveniles: mean=0.76±0.36, median=0.94; singing juveniles: mean=0.74±0.24, median=0.85; singing adults: mean=0.82±0.21, median=0.92), black lines indicate mean values of each group. Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2024 Read the published version in BMC Neuroscience → Version 1 posted Editorial decision: Revision requested 09 Aug, 2024 Editor assigned by journal 26 Jul, 2024 Submission checks completed at journal 25 Jul, 2024 First submitted to journal 23 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4789872","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":337957251,"identity":"347de818-f83d-4386-882d-cd3fe8a8f96a","order_by":0,"name":"Linda Bistere","email":"","orcid":"","institution":"Max Planck Institute for Biological Intelligence","correspondingAuthor":false,"prefix":"","firstName":"Linda","middleName":"","lastName":"Bistere","suffix":""},{"id":337957252,"identity":"b9e4b3a7-42dd-47cd-a265-c6216e19a694","order_by":1,"name":"Stefan Wilczek","email":"","orcid":"","institution":"Max Planck Institute for Biological Intelligence","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Wilczek","suffix":""},{"id":337957253,"identity":"502dd2fa-fc4f-41b7-82bf-1be26c4f9f56","order_by":2,"name":"Daniela Vallentin","email":"data:image/png;base64,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","orcid":"","institution":"Max Planck Institute for Biological Intelligence","correspondingAuthor":true,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Vallentin","suffix":""}],"badges":[],"createdAt":"2024-07-23 15:21:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4789872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4789872/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12868-024-00915-7","type":"published","date":"2024-12-23T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62957244,"identity":"db3d80f8-60bf-45e8-8572-2c34e90739ca","added_by":"auto","created_at":"2024-08-21 12:32:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHVC projection neurons in juvenile birds burst frequently during song production. \u003c/strong\u003eA) Top: example recording of an HVC projection neuron (top: spectrogram of a song motif, below: intracellular recording of an HVC projection neuron during four repetitions of the song motif), Middle: Raster-plot of bursting activity during singing a motif (0-100% of duration) in all birds, Bottom: Probability distribution of burst occurrence during motif. B) Same as in A but for adult birds. C) Maximum number of bursts during song production between juveniles (orange) and adults (grey). D) Number of spikes per trial in juveniles (orange) and adults (grey). E) Distribution of single spikes per trial across all recorded projection neurons in juveniles and adults.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4789872/v1/e3f314577f3195f01f649edc.png"},{"id":62957243,"identity":"9fe0ec13-0c0d-4506-aff1-87beaecc6320","added_by":"auto","created_at":"2024-08-21 12:32:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal dynamics within HVC projection neuron bursts. \u003c/strong\u003eA) Example of a burst in a juvenile during song production (top: spectrogram with a syllable, below: intracellular recording of a single burst), B) same as in A but for an adult bird. C) Number of spikes within a burst in juveniles and adults. D) Distribution of Δ spikes per burst in reoccurring bursts in juveniles and adults. E) Burst duration of all recorded bursts in juveniles and adults. F) Instantaneous firing rate of all bursts in juveniles and adults. G) Progression of instantaneous firing rate in juveniles (left) and adults (right), bold line: mean instantaneous firing rate per number of ISI in juveniles (orange) and adults (grey). H) Demeaned progression of instantaneous firing rate from E) and F) across ISI.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4789872/v1/075266a120ba970f31f312fd.png"},{"id":62957246,"identity":"00bab0b9-1da7-41ea-a199-b2611c23c659","added_by":"auto","created_at":"2024-08-21 12:32:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal features of the subthreshold activity. \u003c/strong\u003eA) Example of excitatory post-synaptic event detection in juveniles (orange) and adults (grey). B) Subthreshold activity exhibited high stereotypy in juveniles and adults. C) Mean PSP duration per HVC projection neuron. D) Distribution of amplitude of all excitatory PSP events recorded. E) Mean frequency of all PSP events per HVC projection neuron.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4789872/v1/3530a57de56359502a9d5823.png"},{"id":72640885,"identity":"97265c49-c2d8-4938-a46f-535700c97163","added_by":"auto","created_at":"2024-12-30 16:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":989020,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4789872/v1/267009b5-24be-4af0-a8c7-68e16ec63e56.pdf"},{"id":62957245,"identity":"2fd5ba0e-8cf4-44d5-8fb4-540517fd95ee","added_by":"auto","created_at":"2024-08-21 12:32:14","extension":"ai","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1521806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1: Subthreshold activity preceding bursts. \u003c/strong\u003eA) Example recording of an HVC projection neuron in a singing juvenile bird (78 days post hatch), aligned to burst onsets across four song motifs. Green lines indicate the selected interval of analysis 15 ms before burst onset. B) Subthreshold activity from the four traces (grey) of the example in A 15 ms window before burst onset. In orange – average subthreshold activity of the 15 ms interval. C) Mean subthreshold stereotypy of all pre-burst intervals per recorded neuron (quiet juveniles: mean=0.76±0.36, median=0.94; singing juveniles: mean=0.74±0.24, median=0.85; singing adults: mean=0.82±0.21, median=0.92), black lines indicate mean values of each group.\u003c/p\u003e","description":"","filename":"SupplFigure1BistereetalBMC.ai","url":"https://assets-eu.researchsquare.com/files/rs-4789872/v1/dc09440232ce0dc46041dbf0.ai"}],"financialInterests":"No competing interests reported.","formattedTitle":"Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe process of acquiring a motor skill encompasses the refinement of a once highly variable movement into its finely-tuned and precise execution. For example, the initial stages of vocal learning in humans are often characterized by inherently variable vocalizations such as cries and babbling sounds\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. After months of practicing these utterances shape into refined words with precise pronunciation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Male zebra finches undergo a comparable learning process for singing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Within a critical period, their initially variable subsong transitions into a plastic song, ultimately culminating in the acquisition of a final, stereotyped song closely resembling that of their tutor\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUsing bioacoustics, the song learning progression of developing zebra finches can be described by assessing changes in spectral features or temporal characteristics of song\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Subsong (~\u0026thinsp;25\u0026ndash;50 days post hatch (dph)) consists of poorly structured sounds with high variability of spectral features (wiener entropy, spectral continuity, pitch and frequency modulation)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Subsong is followed by a plastic song (~\u0026thinsp;50\u0026ndash;80 dph) consisting of structured syllable production that gradually develops distinct acoustic features until a stereotyped, crystalized song is achieved which is characterized by precise, reliably repeated features\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In adults the spectral and temporal features of song are highly stereotyped and acoustic parameters have low variability.\u003c/p\u003e \u003cp\u003eThroughout the developmental phase, neural dynamics in the vocal production pathway undergo various modifications\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, ultimately leading to the emergence of a stable neural pattern during song production\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Initially the lateral magnocellular nucleus of the anterior neostriatum (LMAN) is necessary for the production and timing of subsong vocal patterns\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. LMAN projects to RA (robust nucleus of the arcopallium) which in turn projects to the brainstem vocal and respiratory nuclei\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, leading to audible song production. During the song learning phase, the main input to RA switches from LMAN to HVC, which on a behavioral level is reflected by increased stereotypy in timing and spectral features of song\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. It has been shown to be a gradual and overlapping transition since HVC inactivation during the plastic song phase reverts the juveniles singing into subsong, whereas LMAN inactivation leads to the production of adult like stereotyped song sequences\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe aimed to explore the neural underpinnings that might fine-tune temporal dynamics of plastic song to eventually become a stereotyped song. Between 70 and 90 days post hatch the temporal structure of the song changes i.e. the silent gap duration decreases and the overall timing variability is reduced\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. While RA is necessary for song production throughout all stages of development, its basic spiking characteristics develop concomitantly with spectral features and it does not generate timing\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. LMAN is inducing temporal variability, but only during the subsong phase, since after ~\u0026thinsp;60 dph, the RA motor program is predominantly driven from HVC\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although HVC is not necessary for subsong, it is necessary for all later stages of song production and it has been shown to generate the timing of song in adults\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and juveniles, in which HVC lesions result in the abolishment of adult like gap and syllable durations and HVC cooling results in an elongation of syllables and gaps\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring the early plastic song phase, HVC projection neurons produce rhythmic bursts during several syllables\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. As the song matures, the bursting activity becomes tied to one particular timepoint during song\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, but how these neural changes relate to alterations in the temporal features of singing behavior remains unclear. Here we directly investigated the neural activity changes at a single cell level that underly the transition from plastic song to crystalized song. We quantified the neural dynamics including changes in spiking characteristics and the synaptic inputs generated by connecting neurons by analyzing subthreshold membrane potential in singing zebra finches. Ultimately the crystallized song is sung by adult males to attract females, who prefer a highly temporally precise song.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHVC projection neurons in juveniles exhibit more variable spiking and bursting patterns compared to adults\u003c/h2\u003e \u003cp\u003eThe song development of zebra finches involves a gradual refinement of vocalizations, transitioning from variable juvenile songs to a stable, stereotyped adult form. During the crystallization phase, juveniles begin to produce structured syllables, signaling progress towards their final song. However, the temporal features of their songs, such as the timing between syllables, remain immature and undergo further refinement in the sensorimotor phase\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To understand the neural mechanisms underlying this temporal refinement, we focused on HVC, a premotor nucleus crucial for song learning and production. In adult zebra finches, the HVC motor program for song consists of precise, time-locked bursts of activity\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We aimed to investigate how this program develops during the plastic song phase, a stage of late-stage song learning.\u003c/p\u003e \u003cp\u003eTo achieve this, we conducted intracellular recordings of HVC projection neurons in freely behaving male juvenile zebra finches (74\u0026ndash;94 days post hatch) during singing. Given HVC's role in controlling syllable and gap duration during this phase\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we analyzed the neural activity of HVC projection neurons in both juveniles and adults to identify potential differences that could contribute to the ongoing refinement of song timing.\u003c/p\u003e \u003cp\u003eWe found that HVC projection neurons elicited sparse bursts of action potentials during a specific time point during song production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The distribution of bursts spanned the entire song motif in both age groups\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This observation suggests that the neural dynamics underlying song production are already established during the late phase of song learning, leading to the production of stereotyped song structure. However, this finding alone cannot account for the observed differences in temporal variability between juvenile and adult songs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, indicating that other factors contribute to the ongoing refinement of song timing.\u003c/p\u003e \u003cp\u003eIt has been previously shown that in adult zebra finches HVC projection neurons display a sparse number of bursts during song production\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To explore whether our recorded neurons produced ultra-sparse bursting patterns as well, we calculated the maximum number of bursts per trial for each HVC projection neuron. In juveniles we observed a larger number of bursts during motif per HVC projection neuron when compared to adults (maximum number of bursts per motif: juveniles\u0026thinsp;=\u0026thinsp;0\u0026ndash;4 bursts per motif, median\u0026thinsp;=\u0026thinsp;2, 10 neurons in 4 juveniles; adults\u0026thinsp;=\u0026thinsp;0\u0026ndash;4 bursts per motif, median\u0026thinsp;=\u0026thinsp;1, 54 neurons in 10 adults, p\u0026thinsp;=\u0026thinsp;0.03, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Next, we measured the degree of stereotypy of bursting activity across song motifs. We identified bursts as reoccurring bursts across song motifs, if their onset time was within \u0026plusmn;\u0026thinsp;20ms across motifs. In adults, 85.94% of all recorded bursts were consistently repeated across multiple renditions of the song motif, indicating a high degree of stereotypy. Conversely, in juveniles, only 60% of bursts were reoccurring (number of reoccurring bursts, juveniles\u0026thinsp;=\u0026thinsp;12/20 bursts, adults\u0026thinsp;=\u0026thinsp;55/64 bursts, p\u0026thinsp;=\u0026thinsp;0.02, Fisher Exact Test). These observations are in line with previous work\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and suggest, that the functional connectivity within HVC exhibits greater variability in late-stage juveniles compared to adults.\u003c/p\u003e \u003cp\u003eIn addition to bursting activity some neurons also exhibited single action potentials during song production which reduces the sparseness of the neural code and might induce less reliable behavioral outcomes in terms of song consistency and timing. To test whether single action potentials might contribute to a less temporally stereotyped song we explored the spiking activity of the HVC neurons (spikes within a burst and single spikes). We observed, that the number of spikes per motif tended to be higher in juvenile birds than in adults despite not significantly so (spikes per motif: juveniles\u0026thinsp;=\u0026thinsp;6.5 spikes/motif, adults\u0026thinsp;=\u0026thinsp;4 spikes/motif, p\u0026thinsp;=\u0026thinsp;0.08, one-way ANOVA, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Higher number of spikes per motif could also be attributed to single spikes alone outside of bursts. To account for the potential impact of single spikes, we separately analyzed their occurrence across song renditions. This analysis revealed that HVC neurons in juvenile zebra finches exhibited a significantly higher number of single action potentials compared to adults (single spikes per motif: juveniles\u0026thinsp;=\u0026thinsp;0.25 single spikes/motif, adults\u0026thinsp;=\u0026thinsp;0 single spikes/motif, p\u0026thinsp;=\u0026thinsp;0.03, one-way ANOVA, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The number of spikes recruited for bursting activity per song motif did not differ between juveniles and adults on a single neuron level (number of spikes within bursts: juveniles\u0026thinsp;=\u0026thinsp;5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54, median\u0026thinsp;=\u0026thinsp;5.25, adults\u0026thinsp;=\u0026thinsp;3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96, median\u0026thinsp;=\u0026thinsp;3.75, p\u0026thinsp;=\u0026thinsp;0.23, one-way ANOVA, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results show that in late-stage development HVC projection neurons in juveniles exhibit detectable differences in spiking activity compared to adults. However, this increased spiking activity in juveniles is not reflected in the spiking patterns within bursts themselves. Instead, it can be attributed to a higher frequency of single spikes occurring outside of bursts. This increased incidence of single spikes might contribute to the greater variability observed in juvenile song production. We next explored, whether the song variability in juveniles during the late-state development can also be attributed to temporal dynamics within bursts in HVC projection neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTemporal dynamics of HVC neuron bursting activity are slower in juveniles compared to adults\u003c/h2\u003e \u003cp\u003eWe explored the intrinsic dynamics of individual bursts by analyzing their individual number of spikes and temporal characteristics of these spikes during song production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). First, we quantified the number of spikes occurring per burst. In both juveniles and adults, we observed a similar number of spikes occurring per burst (median spikes per burst\u0026thinsp;\u0026plusmn;\u0026thinsp;std: juveniles\u0026thinsp;=\u0026thinsp;3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95, adults\u0026thinsp;=\u0026thinsp;3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94, p\u0026thinsp;=\u0026thinsp;0.26, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Next, we assessed the variability of the number of spikes occurring in each reoccurring burst. HVC projection neurons in juveniles had a comparable distribution of Δ number of spikes per burst to neurons in adults (Δ number of spikes per burst: juveniles\u0026thinsp;=\u0026thinsp;0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61, adults\u0026thinsp;=\u0026thinsp;0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29, p\u0026thinsp;=\u0026thinsp;0.09, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), which indicates stereotyped reoccurring bursts during the late-stage development. We next investigated whether the temporal structure of bursts in juveniles and adults differed by quantifying their duration. Unlike previously reported\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we did not observe differences in the duration of bursts between juveniles and adults on a population level potentially due to our smaller sample size (duration of bursts: juveniles\u0026thinsp;=\u0026thinsp;9.64 ms, adults\u0026thinsp;=\u0026thinsp;6.83 ms, Wilcoxon rank sum test, p\u0026thinsp;=\u0026thinsp;0.15, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). However, when assessing the instantaneous firing rate within bursts, we found that it was lower in juveniles than adults (firing rate: juveniles\u0026thinsp;=\u0026thinsp;226.09 Hz, adults\u0026thinsp;=\u0026thinsp;387.68 Hz, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) indicating that premotor signals are produced on a slower timescale compared to in the adult brain. To quantify whether the lower instantaneous firing rate also produced a distinct temporal pattern of spiking progression within bursts, we next compared the normalized pattern of spiking progression in juveniles and adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Once we accounted for the overall higher instantaneous firing rate in adults by demeaning the progression pattern in juveniles and adults, the relative progression in juveniles was not distinguishable from that of the adult HVC projection neurons (demeaned firing rate: juveniles\u0026thinsp;=\u0026thinsp;3.41*10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e\u0026plusmn;25.45 Hz, adults=-1.14*10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e\u0026plusmn;55.04 Hz, p\u0026thinsp;=\u0026thinsp;0.84, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eTo verify, whether the synaptically connected neurons provide stereotyped excitatory inputs preceding the bursts, we analyzed the membrane potential rise during a 15 ms window preceding all recorded bursts. Excitatory input accounting to the membrane potential rise preceding the bursts was as stereotyped in singing juveniles as in quiet juveniles where we elicited bursts using current injection (Wilcoxon rank sum test, p\u0026thinsp;=\u0026thinsp;0.21) or in singing adults (Wilcoxon rank sum test, p\u0026thinsp;=\u0026thinsp;0.11, Supplementary Fig.\u0026nbsp;1). This finding indicates, that the elicitation of bursts in singing juveniles occurs in a stereotyped way that is comparable to adults. The overall stereotyped characteristics of bursts (i.e., number of spikes, burst duration and stereotyped excitatory input) and the lower instantaneous firing rate indicate precise yet slower signal transmission in juvenile HVC projection neurons than in adults. We hypothesized, that these temporal dynamics might also be exhibited in a focal microcircuit within HVC. Here we leveraged the ability to quantify subthreshold activity of individual HVC projection neurons. This metric reflects the integrated input received by a focal neuron from its presynaptic network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHVC projection neurons receive temporally distinct subthreshold inputs in juveniles compared to in adults\u003c/h2\u003e \u003cp\u003eTo quantify the temporal dynamics of inputs that HVC projection neurons are receiving, we assessed the stereotypy of the membrane potential by correlating subthreshold activity across motif renditions for each recorded neuron (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In juveniles, the subthreshold activity was as stereotyped as in adults (subthreshold precision: juveniles\u0026thinsp;=\u0026thinsp;0.82, adults\u0026thinsp;=\u0026thinsp;0.83, p\u0026thinsp;=\u0026thinsp;0.59, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting a stable neural representation of song production. Since we previously reported temporal differences in bursting activity, we hypothesized, that these differences could also be reflected in the summation of excitatory and inhibitory postsynaptic potentials (PSPs) received by a focal neuron. To address these temporal dynamics, we quantified the duration of the PSPs and observed, that PSP events in juveniles were of longer duration than in adults (PSP duration: juveniles\u0026thinsp;=\u0026thinsp;21.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94, adults\u0026thinsp;=\u0026thinsp;18.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54, p\u0026thinsp;=\u0026thinsp;0.01, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), which is in line with our previously observed slower temporal dynamic within bursts in juveniles. Further we quantified the membrane potential amplitude, and found that PSPs in juveniles had a higher membrane potential amplitude than in adults (PSP amplitude: juveniles\u0026thinsp;=\u0026thinsp;6.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2mV, adults\u0026thinsp;=\u0026thinsp;5.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), which could potentially be accounted to a higher resting membrane potential in juveniles\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In juveniles, a higher resting membrane potential is associated with altered neuronal excitability and could likely manifest as changes in the frequency or amplitude of postsynaptic potential events. The frequency of the PSP element occurrence was lower in juveniles than adults (PSP element frequency: juveniles\u0026thinsp;=\u0026thinsp;12.85\u0026thinsp;\u0026plusmn;\u0026thinsp;4.99, adults\u0026thinsp;=\u0026thinsp;16.03\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91, p\u0026thinsp;=\u0026thinsp;0.045, Wilcoxon rank sum test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), suggesting a slower-paced, more sparse input from synaptically connected neurons. The slower and more sparse membrane potential dynamics during singing in juveniles might explain the temporal differences during song performance\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we leveraged the developmental transition in song production\u0026mdash;from the variable plastic song of juvenile zebra finches to the crystallized song of adults\u0026mdash;to investigate the neural mechanisms underlying this behavioral shift. We focused specifically on the HVC, a premotor nucleus critical for song learning and production, to understand the neural dynamics that facilitate this transformation. The song crystallization phase in late-stage juveniles is characterized by a higher number of bursts of HVC projection neurons during singing, that were more variable in their occurrence than bursts of HVC projection neurons in adult birds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Such neural pattern may underlie the more variable and exploratory vocalizations observed in juveniles during song learning. Despite receiving stereotyped excitatory input, bursts in juveniles exhibited a lower instantaneous firing rate than in adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding suggests that developmental changes in intrinsic neuronal properties may play a role in how the temporal information is encoded within HVC. Further, the stereotyped post-synaptic events occurring at a lower frequency in juveniles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) may indicate slower dynamics in their excitatory drive.\u003c/p\u003e \u003cp\u003eIn adult birds, HVC-RA projection neurons burst sparsely during singing\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These bursts are triggered by presynaptic inputs with a range of latencies to form neural sequences during song production\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In juvenile birds, the connectivity in HVC develops over time by bursting events gradually becoming more timepoint-specific during singing\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In this study, we report that the bursting activity in the late-stage juveniles occurred more frequently with less stereotypy across motifs than in adults. The development of song in zebra finches is accompanied by a progressive increase in inhibition within the HVC\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In adult birds, the precise activity of inhibitory neurons is crucial for shaping the sparse bursts of excitatory neurons within HVC, which are thought to contribute to the stereotyped nature of adult song\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It is plausible that the excitatory-inhibitory balance within the HVC of the juvenile birds we recorded from was not yet fully matured, potentially contributing to the observed differences in bursting activity compared to adults.\u003c/p\u003e \u003cp\u003eIn terms of subthreshold activity, neural dynamics were stable during song production in both juveniles and adults, which is in line with a previous study reporting highly stereotyped song performance of female-directed songs in juveniles, that were more variable during song practice\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Additionally, the higher amplitude post-synaptic potentials in juveniles could be another indicator of lower inhibitory current within HVC during song performance. Alternatively, neural dynamics may also have distinct temporal signatures due to different membrane properties of the HVC projection neurons in juveniles and adults. During development, the membrane properties of HVC projection neurons exhibit a higher resting membrane potential and an increased spiking amplitude\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, which could lead to a higher general excitability of the HVC projection neurons. In line with this hypothesis, we report an increased amplitude of the excitatory post-synaptic events in juveniles. These excitatory post-synaptic events occurred at a lower frequency during singing in juveniles compared to in adults. The slower temporal dynamics could be attributed to incomplete myelination of axonal connections and changing membrane properties during development\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, that ultimately impact the signal propagation within HVC during the juvenile stage. In line with this hypothesis, studies in adults demonstrate relatively slow HVC projection neuron conduction velocities\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, intrinsic properties of a subset of HVC projection neurons exhibit higher variability during song learning than in adulthood\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, likely leading to a higher variability in the neural dynamics within HVC during song production. Investigating the signal propagation within HVC during development would yield more insights as to whether it also plays a role into HVC network dynamics during song learning.\u003c/p\u003e \u003cp\u003eWe revealed distinct temporal dynamics within HVC during the song crystallization phase, where temporal and spectral features of the song are being fine-tuned. Despite the differences in temporal dynamics of HVC projection neurons during the song crystallization phase and adulthood, the overall stereotypy of neural patterns during song production remained stable. However, the observed differences in spiking and subthreshold activity between juvenile and adult HVC projection neurons underscore the dynamic nature of neural circuits during song learning. This suggests that HVC undergoes significant plasticity during development to establish the stable, stereotyped patterns associated with mature song production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal housing\u003c/h2\u003e \u003cp\u003e All procedures were approved by the Regierungspr\u0026auml;sidium Oberbayern (VET 02-21-201), Landesamt f\u0026uuml;r Gesundheit und Soziales (LAGeSo Berlin) (G 0225/16) at the Freie Universit\u0026auml;t Berlin or according to Institutional Animal Care and Use Committee at New York University Langone Medical Center guidelines. Juvenile zebra finches (n\u0026thinsp;=\u0026thinsp;4, older than 73 dph) were housed in an aviary or a breeding cage with their genetic parents up to 60 dph, at which they were moved to an adjacent aviary where they had visual and auditory contact with their parents. Adult male birds (n\u0026thinsp;=\u0026thinsp;10, older than 100 dph) were acquired from a breeder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSurgery\u003c/h2\u003e \u003cp\u003eMale zebra finches were anesthetized with isoflurane (concentration: 1\u0026ndash;3% isoflurane, 97\u0026ndash;99% oxygen) at a 65\u0026deg; head angle. An incision was made to expose the skull and a square shaped area of trabecular bone was removed above HVC, RA and cerebellum using a dental drill (carbide bur, FG \u0026frac14;, Johnson-Promident). Nucleus RA was targeted according to coordinates (0 point at the bifurcation of the midsaggital sinus, RA coordinates: posterior 1.85 mm, lateral 2.25 mm, ventral 1.8 mm). A carbon fiber electrode (Kation Scientific, LLC) was used to identify RA based on the firing pattern \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Next, we targeted HVC using coordinates (0 point at the bifurcation of the midsaggital sinus, HVC coordinates: anterior 0.2 mm, lateral 2.3 mm, ventral 0.2 mm) and confirmed the location with antidromic stimulation from RA \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A previously assembled microdrive for intracellular recordings\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e was then implanted at 25\u0026deg; angle above HVC for intracellular recordings. Birds were let to recover 1\u0026ndash;3 days before the intracellular recordings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular recordings in freely behaving animals\u003c/h2\u003e \u003cp\u003eTo record intracellularly we used sharp intracellular electrodes (borosilicate glass with filament, 0.1 mm diameter), that were previously pulled using a micropipette puller (Model P-97, Sutter Instrument). Before use, each electrode was backfilled with potassium acetate (concentration: 3M). Using a silicone elastomer, we built a well around the craniotomy in HVC and filled it with phosphate buffered saline (PBS). Next, we removed dura using a dura pick. While birds were in a head-fixed setting, we lowered down the glass electrode in HVC until we could identify surrounding cells using oscilloscope and an audio monitor. We then transferred the bird into the cage of the recording setup and gradually lowered the electrode within HVC, accompanied by a brief buying pulse (10\u0026ndash;20 ms), until a successful penetration of a neuron. Next, we presented a female bird to the male bird to motivate singing behavior. For this study, we only selected neurons that had at least 30 mV action potentials, the recording lasted at least 3 minutes and the membrane potential was below \u0026minus;\u0026thinsp;50 mV. We could identify HVC projection neurons based on their low firing rate\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and characteristic waveform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSong acquisition\u003c/h2\u003e \u003cp\u003eWe briefly housed the juvenile and adult birds in sound attenuated boxes together with a female to motivate singing. Once song was recorded, we took one motif from each bird and detected syllables and gaps using threshold-based detection from the sound envelope. Durations of each segment were calculated by subtracting onset timing from the offset timing respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSpike and burst detection\u003c/h2\u003e \u003cp\u003eSpikes in each recorded trace were detected as events exceeding the 15 mV threshold from the demeaned trace. We defined bursts as single events where the firing rate of adjacent spikes exceeded 100 Hz and the spikes occurred on the same membrane depolarization event. To compare reoccurring bursts, we defined a burst as reoccurring, if the onset time of the burst (first spike) in next trial occurred\u0026thinsp;\u0026plusmn;\u0026thinsp;20 ms from the onset of burst in the previous trial. Delta of spikes per burst was calculated for each identified, reoccurring burst. First, the mode of spikes per burst was calculated for each reoccurring burst. Then, the number of spikes of each burst of the same identity was subtracted from mode and an absolute mean delta of number of spikes was calculated per identified burst.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInstantaneous firing rate\u003c/h2\u003e \u003cp\u003eInstantaneous firing rate was calculated as the mean firing rate per reoccurring burst and was defined as number of inter spike interval (ISI) divided by the sum of ISIs per burst. To make a fair comparison of the temporal progression of spiking within bursts, we only considered bursts with no more than 6 spikes per burst for this analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExcitatory post-synaptic potential detection\u003c/h2\u003e \u003cp\u003eFor detection of the PSPs, we first detected local peaks and events of membrane depolarization exceeding 2 mV amplitude, then measured their amplitude and calculated the frequency by dividing the number of events with the duration of the song motif. For the analysis of PSP duration, we only included PSPs that were shorter than 35 ms to avoid false PSP duration detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSubthreshold stereotypy\u003c/h2\u003e \u003cp\u003eSubthreshold stereotypy was defined as a cross correlation at 0 lag of all trials within a cell. First, we cut the spikes of our recorded traces (spike detection as described above). We then demeaned all subthreshold traces for a fair comparison. Each trace was then correlated with every other trace from the same HVC projection neuron.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eL.B. and D.V. conceived the study and designed the experiments. L.B. and S.W. conducted the experiments. L.B. analyzed the data. L.B. and D.V. wrote the manuscript, D.V. acquired funding and supervised the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement:\u003c/strong\u003e No competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate:\u003c/strong\u003e All procedures were approved by the Regierungspr\u0026auml;sidium Oberbayern (VET 02-21-201), Landesamt f\u0026uuml;r Gesundheit und Soziales (LAGeSo Berlin) (G 0225/16) at the Freie Universit\u0026auml;t Berlin or according to Institutional Animal Care and Use Committee at New York University Langone Medical Center guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication:\u003c/strong\u003e\u0026nbsp; The authors hereby give our consent for the publication of the manuscript titled \u0026quot;Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches\u0026rdquo; in BMC Neuroscience We confirm that we have had the opportunity to review the manuscript and understand that it will be made publicly available upon publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the HORIZON EUROPE European Research Council (ERC)-2017-StG-757459 MIDNIGHT, the Deutsche Forschungsgemeinschaft VA742/2-1 and the Deutsche Forschungsgemeinschaft 327654276\u0026ndash;SFB 1315\u0026mdash;awarded to D.V.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eWe would like to thank the guest editors Jasmine L. Loveland and Bradley M. Colquitt for inviting us to contribute to the Collection \u0026lsquo;Behavioral neuroscience of vocal learning in avian and mammalian species\u0026rsquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOller, D. K. Chapter 6 - THE EMERGENCE OF THE SOUNDS OF SPEECH IN INFANCY. in \u003cem\u003eChild Phonology\u003c/em\u003e (eds. Yeni-komshian, G. H., Kavanagh, J. F. \u0026amp; Ferguson, C. A.) 93\u0026ndash;112 (Academic Press, 1980). doi:10.1016/B978-0-12-770601-6.50011-5.\u003c/li\u003e\n\u003cli\u003eLightfoot, D. Language acquisition and language change. \u003cem\u003eWIREs Cognitive Science\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 677\u0026ndash;684 (2010).\u003c/li\u003e\n\u003cli\u003eDoupe, A. J. \u0026amp; Kuhl, P. K. \u003cem\u003eBIRDSONG AND HUMAN SPEECH: Common Themes and Mechanisms\u003c/em\u003e. www.annualreviews.org (1998).\u003c/li\u003e\n\u003cli\u003eTchernichovski, O., Mitra, P. 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Long-range inhibition within the zebra finch song nucleus RA can coordinate the firing of multiple projection neurons. \u003cem\u003eJournal of Neurophysiology\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 3007\u0026ndash;3020 (1999).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"vocal learning, HVC, songbirds","lastPublishedDoi":"10.21203/rs.3.rs-4789872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4789872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZebra finches undergo a gradual refinement of their vocalizations, transitioning from variable juvenile songs to the stereotyped song of adulthood. To investigate the neural mechanisms underlying song crystallization, a critical phase in this developmental process, we conducted intracellular recordings in HVC, a premotor nucleus essential for song learning and production. We found that HVC projection neurons in juvenile zebra finches in the song crystallization phase exhibited more variable spiking patterns compared to the precise bursting observed in adult HVC projection neurons. Additionally, subthreshold membrane potential fluctuations in juvenile neurons were characterized by longer duration and larger amplitude excitatory postsynaptic potentials. These distinct temporal dynamics in HVC during song crystallization likely play a crucial role in the fine-tuning processes that shape the precise timing and structure of the mature zebra finch song.\u003c/p\u003e","manuscriptTitle":"Variable and slow-paced neural dynamics in HVC underlie plastic song production in juvenile zebra finches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 12:32:10","doi":"10.21203/rs.3.rs-4789872/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-09T04:53:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T09:30:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-25T12:13:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Neuroscience","date":"2024-07-23T15:19:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96036000-ced9-459a-89f5-c6677d79f138","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:05:10+00:00","versionOfRecord":{"articleIdentity":"rs-4789872","link":"https://doi.org/10.1186/s12868-024-00915-7","journal":{"identity":"bmc-neuroscience","isVorOnly":false,"title":"BMC Neuroscience"},"publishedOn":"2024-12-23 15:57:36","publishedOnDateReadable":"December 23rd, 2024"},"versionCreatedAt":"2024-08-21 12:32:10","video":"","vorDoi":"10.1186/s12868-024-00915-7","vorDoiUrl":"https://doi.org/10.1186/s12868-024-00915-7","workflowStages":[]},"version":"v1","identity":"rs-4789872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4789872","identity":"rs-4789872","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}