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Thalamus drives vocal onsets in the zebra finch courtship song

Nature volume 616, pages 132–136 (2023)Cite this article

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Abstract

While motor cortical circuits contain information related to specific movement parameters1, long-range inputs also have a critical role in action execution2,3. Thalamic projections can shape premotor activity2,3,4,5,6 and have been suggested7 to mediate the selection of short, stereotyped actions comprising more complex behaviours8. However, the mechanisms by which thalamus interacts with motor cortical circuits to execute such movement sequences remain unknown. Here we find that thalamic drive engages a specific subpopulation of premotor neurons within the zebra finch song nucleus HVC (proper name) and that these inputs are critical for the progression between vocal motor elements (that is, ‘syllables’). In vivo two-photon imaging of thalamic axons in HVC showed robust song-related activity, and online perturbations of thalamic function caused song to be truncated at syllable boundaries. We used thalamic stimulation to identify a sparse set of thalamically driven neurons within HVC, representing ~15% of the premotor neurons within that network. Unexpectedly, this population of putative thalamorecipient neurons is robustly active immediately preceding syllable onset, leading to the possibility that thalamic input can initiate individual song components through selectively targeting these ‘starter cells’. Our findings highlight the motor thalamus as a director of cortical dynamics in the context of an ethologically relevant behavioural sequence.

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Fig. 1: Uva perturbations impact normal syllabic song structure.
Fig. 2: Structure and function of thalamic inputs to HVC.
Fig. 3: Classifying HVC premotor neurons on the basis of thalamic drive.
Fig. 4: Differential roles of UvD and nUvD neurons during song production.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank A. Banerjee, D. Jin, N. Nikbakht, A. Nieder, L. Veit, C. Bischof and members of the Long laboratory for comments on earlier versions of this manuscript. We thank J. Moore for technical advice on fluorescence quantification. A. Paulson provided technical assistance. This research was supported by R01 NS075044 (M.A.L.), F31 NS116933 (M.E.) and Simons Collaboration on the Global Brain (M.A.L.).

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Authors and Affiliations

  1. NYU Neuroscience Institute and Department of Otolaryngology, New York University Langone Medical Center, New York, NY, USA

    Felix W. Moll, Devorah Kranz, Ariadna Corredera Asensio, Margot Elmaleh, Lyn A. Ackert-Smith & Michael A. Long

  2. Center for Neural Science, New York University, New York, NY, USA

    Felix W. Moll, Devorah Kranz, Ariadna Corredera Asensio, Margot Elmaleh, Lyn A. Ackert-Smith & Michael A. Long

  3. Animal Physiology, Institute of Neurobiology, University of Tübingen, Tübingen, Germany

    Felix W. Moll

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Contributions

F.W.M. and M.A.L. conceived the study and designed the experiments; F.W.M., M.E., L.A.A.-S., D.K. and A.C.A. conducted the research; F.W.M., M.E., L.A.A.-S. and M.A.L. performed data analyses; F.W.M., L.A.A.-S., A.C.A. and M.A.L. created the figures; F.W.M. and M.A.L. wrote the initial draft of the manuscript; F.W.M., M.E., A.C.A. and M.A.L. edited and reviewed the final manuscript. M.E. and M.A.L. acquired funding; M.A.L. supervised the project.

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Correspondence to Michael A. Long.

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Extended data figures and tables

Extended Data Fig. 1 Uva lesions impact syllabic song structure.

a,b, Silhouette criteria calculated on the prelesion (a) and postlesion (b) vocalizations for each bird (n = 8) for k-means of various sizes embedded using the first five PCs. Black circles superimposed on curves indicate the number of syllables in each bird’s song motif. c,d, Histology of Uva lesioned (n = 6) (c) and sham lesioned (n = 2) (d) birds (brightfield images, sagittal slices). White arrows indicate the extent of the lesions. CP: Posterior commissure, OM: occipitomesencephalic tract, SpM: nucleus spiriformis medialis, Cb: cerebellum. e, Schematic of the zebra finch brain at the medio-lateral level of Uva (sagittal view) based on an established anatomical resource (http://www.zebrafinchatlas.org/). The highlighted region is used in (f) and (g) to show the lesion extent (in transparent blue) for individual birds. f, g, t-SNE plots representing vocalizations taken from Uva lesioned (f) and sham lesioned (g) birds (prelesion green, postlesion black). Example sonograms depict pre- and postlesion song motifs.

Extended Data Fig. 2 Circuit-level consequences of electrical stimulation.

a,b, Example spike rasters (top) and peristimulus time histograms (bottom) for 6 HVC neurons that respond transiently (a) or are suppressed (b) by local high-frequency stimulation (see Methods). Red line indicates the onset of stimulation. Intensity and timing of stimulation identical to that used in behavioral experiments (i.e., Fig. 1). Inset: Average waveform for each neuron shows a variety of spike widths (Spike rise times - Cell a: 0.09 ms, Cell b: 0.34 ms, Cell c: 0.20 ms, Cell i: 0.36 ms, Cell ii: 0.41 ms, Cell iii: 0.30 ms. c, Activity profiles of 64 neurons recorded during local stimulation from 2 birds (bin size: 25 ms). At right, a closer view of responses from transiently active neurons (bin size: 5 ms). Color scale ranges from 0 (no firing) to 1 (maximum firing rate during observation period). d, Histogram showing the spike rise times (see Methods) for all recorded neurons. Transiently active neurons (red) often displayed shorter rise times, indicative of inhibitory interneurons.

Extended Data Fig. 3 Stimulation-related song truncation times across all birds.

a, Histology was used to confirm the placement of our stimulating electrodes (n = 10 birds). Shown here is an example bird in which we labeled upstream nuclei labeled with a retrograde GFP virus into HVC (n = 3 birds), including nucleus Uvaeformis (Uva) and nucleus interfacialis of the nidopallium (NIf). Arrows indicate the bipolar electrode tract (left) and the axonal tract emanating from Uva (right). b, Photography of bipolar stimulation electrode. c,d, Magnified views of Uva from the bird shown in (a). In (d), the dotted white line indicates the electrode tract and the diffuse green fluorescence nearby are small electrolytic lesions made to identify the electrode site. e–k, Stimulation (black ticks) and song truncation times (Uva: red ticks; HVC: blue ticks) for all stimulated motifs aligned to sonogram shown on top (E: 325 trials, F: 119 trials, G: 114 trials, H: 187 trials, I: 331 trials, J: 163 trials, K: 214 trials). For each plot, a histogram (bin size: 10 ms) shows the probability of a stimulation event and song truncation to occur (stimulation = black, Uva truncation: red, HVC truncation: blue). Orange bars indicate examples of long duration, complex syllables. l, Example songs truncated by unilateral Uva stimulation in either the left or the right hemisphere (yellow lines = stimulation times, red lines = song truncation times). Reference song at top. m, Proportion of song truncations related to closest syllable offset for unilateral stimulation (bird 156, left hemisphere: 86 trials; bird 156, right hemisphere: 64 trials; bird 471, right hemisphere: 32 trials).

Extended Data Fig. 4 Quantifying the population of HVC-projecting Uva neurons.

a–c, Sagittal view of HVC (white dotted line) into which retrograde tracers were injected (right is anterior) (n = 3 birds). NeuN labeled neurons shown in orange (a). Extent of fluorescently labeled cholera toxin subunit B (red) relative to HVC (b). Local labeling of cells with EGFP (green) after the injection of a retrograde virus into HVC (c). d–f, Spheres indicate retrogradely labeled HVC-projecting Uva neurons from three zebra finches shown in parasagittal (d), coronal (e), and transverse (f) views. Scale bars: 200 µm.

Extended Data Fig. 5 Uva-stimulation responses in each HVC cell type.

a–c, Activation ratios following Uva stimulation for premotor neurons (HVCRA) (a), basal ganglia-projecting neurons (HVCX) (b), and putative interneurons (HVCInt) and Avalanche-projecting neurons (HVCAv) (c). Left histogram same data as plots at right but on a logarithmic axis. d, Individual (gray) and average (red) fluorescence changes following Uva stimulation for each Uva driven HVCRA neuron from Fig. 3g.

Extended Data Fig. 6 UvD and nUvD spiking event times relative to song for premotor neurons across all birds.

a–f, All spiking event times for UvD and nUvD neurons across all birds aligned to sonogram and amplitude of song motif. Bird 490 (UvD = 11, nUvD = 58) (a), Bird 471 (UvD = 1, nUvD = 13) (b), Bird 513 (UvD = 10, nUvD = 30) (c), Bird 520 (UvD = 6, nUvD = 51) (d), Bird 486 (UvD = 2, nUvD = 1) (e), Bird 467 (UvD = 0, nUvD = 21) (f). g, Comparison of song amplitude following UvD and nUvD spiking events. nUvD (black) and UvD (red) lines show the median song amplitude following spiking events for recorded data (window: 4 to 19 ms post spiking event; nUvD, n = 174, UvD, n = 30) compared with a 99% confidence interval (CI) based on the distribution of 100,000 median song amplitude values (same window) drawn from data matched numbers of uniformly distributed, random event times (see Methods).

Extended Data Fig. 7 Spiking event times of UvD and nUvD HVCRA and HVCX neurons aligned to vocalization onset and offset.

a, Spiking event times of HVCRA neurons active during singing. Left: Aligned to the closest syllable onset (UvD, n = 23; nUvD, n = 106). Right: Aligned to the closest syllable offset (UvD, n = 19; nUvD, n = 89). b, Spiking event times of HVCRA neurons active during introductory notes, distance calls, or tet calls. Left: Aligned to the closest vocalization onset (UvD, n = 8, nUvD, n = 19). Right: Aligned to the closest vocalization offset (UvD, n = 0, nUvD n = 11). No spiking events from UvD HVCRA neurons were found within 50 ms of introductory note or call offsets. c, Left: Scatterplot displaying activation ratios and Uva stimulation evoked ΔF/F for 227 HVCRA neurons observed in the ketamine condition. Filled circles in the UvD cluster (red) were also classified as UvD cells in the awake condition shown in (d). Middle: Spiking event times of HVCRA neurons – classified based on the ketamine condition – aligned to the closest syllable onset (UvD, n = 29; nUvD, n = 114). Right: Aligned to the closest syllable offset (UvD, n = 18; nUvD, n = 90). d, Left: Same as (c) for 220 HVCRA neurons observed in the awake condition. Filled circles in the UvD cluster (red) were also classified as UvD cells in the ketamine condition shown in (c). Middle: Spiking event times of HVCRA neurons – classified based on the awake condition – aligned to the closest syllable onset (UvD, n = 24; nUvD, n = 113). Right: Aligned to the closest syllable offset (UvD, n = 13; nUvD, n = 88). e, Left: Scatterplot displaying activation ratios and Uva stimulation evoked ΔF/F for 667 HVCX neurons. Neurons were classified using either k-means clustering (top) or a ‘strict’ activation ratio threshold (bottom), which was set to 0.38 (i.e. the lowest activation ratio of the UvD cluster in Fig. 3g). Middle: Spiking event times of UvD HVC neurons aligned to the closest syllable onset (k-means: HVCRA, n = 31; HVCX, n = 50; strict: HVCX, n = 28). Right: Aligned to the closest syllable offset (k-means: HVCRA, n = 19; HVCX, n = 23, strict: HVCX, n = 13). Horizontal red, green or blue lines indicate moments in which the population exceeds a 99% CI of a uniform distribution.

Extended Data Fig. 8 Uva driven (UvD) premotor neurons active at motif and bout transition points.

a, Average fluorescence responses (± SEM) to Uva stimulation for 5 functionally defined UvD HVC neurons. Range: 23–26 stimulation trials. b, Activity of UvD neurons during singing. Song amplitude shown at top; fluorescence trace in red (scale bar = 2 ΔF/F). Individual song motifs indicated by green background shading. c, Song motif aligned fluorescence responses for all songs during which Cell #1 (n = 42 trials), Cell #70 (n = 15 trials), Cell #8 (n = 65 trials), Cell # 85 (n = 23 trials), and Cell #117 (n = 24 trials) were recorded. Black dotted line indicates the median spiking event time estimated across all fluorescence traces (see Methods). Traces normalized by maximum value.

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Supplementary Video 1

HVC activity during song. Imaged neurons expressing GCaMP6 show consistent activity during the performance of six repeated song motifs. Reponses are shown in real time. Neurons projecting to Area X are labelled with a red retrograde tracer (Methods).

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Moll, F.W., Kranz, D., Corredera Asensio, A. et al. Thalamus drives vocal onsets in the zebra finch courtship song. Nature 616, 132–136 (2023). https://doi.org/10.1038/s41586-023-05818-x

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