Identification of a motor-to-auditory pathway important for vocal learning

Abstract

Learning to vocalize depends on the ability to adaptively modify the temporal and spectral features of vocal elements. Neurons that convey motor-related signals to the auditory system are theorized to facilitate vocal learning, but the identity and function of such neurons remain unknown. Here we identify a previously unknown neuron type in the songbird brain that transmits vocal motor signals to the auditory cortex. Genetically ablating these neurons in juveniles disrupted their ability to imitate features of an adult tutor's song. Ablating these neurons in adults had little effect on previously learned songs but interfered with their ability to adaptively modify the duration of vocal elements and largely prevented the degradation of songs' temporal features that is normally caused by deafening. These findings identify a motor to auditory circuit essential to vocal imitation and to the adaptive modification of vocal timing.

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Figure 1: A distinct class of projection neurons links the song premotor nucleus HVC to the auditory region Av.
Figure 2: HVCAv neurons transmit motor-related signals during song production.
Figure 3: HVCAv neurons receive selective input from premotor HVCRA neurons.
Figure 4: Intersectional genetic ablation of HVCAv neurons in juvenile zebra finches impairs their ability to copy a tutor song.
Figure 5: Intersectional ablation of HVCAv neurons in adult birds does not disrupt song production but does attenuate deafening-induced degradation of song's temporal features.
Figure 6: Intersectional ablation of HVCAv neurons in adult zebra finches interferes with feedback-dependent plasticity of song element timing but not syllable pitch.

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Acknowledgements

The authors thank D. Schneider, K. Tschida, T. Warren and S. Lisberger for reading and commenting on the manuscript; K. Hamaguchi (Duke University Medical Center) for software support; W.Y. Peh (Duke University Medical Center) for help with calcium imaging experiments; B.P. Olveczky (Harvard University) for providing Conditional Auditory Feedback software; J. Baltzegar, M. Booze (Duke University Medical Center), J. Holdway and A. Guerrero (UT Southwestern Medical Center) for animal husbandry and laboratory support. This research was supported by grants from the National Science Foundation (R.M. (IOS-1354962), T.F.R. (IOS-1457206, IOS-1451034)), the US National Institutes of Health (R.M. (R01DC002524, R01NS099288), T.F.R. (R01DC014364), N.M.S. (R01NS049488, R01NS083872), M.J.K. (F30NS096871)), an Inscopix DECODE Award (R.M.), the Klingenstein-Simons Fellowship (T.F.R.), the Ellison Medical Foundation (N.M.S.), a JSPS Postdoctoral Fellowship for Research Abroad (M.T.), an Alpha Omega Alpha Research Fellowship (G.C.) and a NARSAD Young Investigator Grant (Essel Investigator) from the Brain & Behavior Research Foundation (T.F.R.).

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Contributions

T.F.R., E.H. and R.M. conceived and designed all experiments. T.F.R. did the anatomical tracing experiments and juvenile genetic lesion experiments, and designed and oversaw the conditional auditory feedback experiments. E.H. did the anatomical tracing experiments, adult genetic lesioning experiments and adult deafening experiments. M.T., M.G.K. and G.C. did the slice physiology, calcium imaging and conditional auditory feedback experiments, respectively. C.F.Y. and N.M.S. provided virus used for cell ablation experiments. T.F.R. and R.M. wrote the manuscript. All authors read and commented on the manuscript.

Corresponding authors

Correspondence to Todd F Roberts or Richard Mooney.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparisons of GFP-expressing HVCAv, HVCRA and HVCX cell bodies.

a) Soma diameter measured at the long axis of the cell body for HVCAv (green), HVCRA (red), and HVCX (blue) cells (two-tailed t-test: HVCAv versus HVCRA, n= 29 for both groups, p = 0.0001; HVCAv versus HVCX, n = 29 for both groups, p = 0.0006). b) Soma area for HVCAv (green), HVCRA (red), and HVCX (blue) cells (two-tailed t-test: HVCAv versus HVCRA, n= 29 for both groups, p = 0.229; HVCAv versus HVCX, n = 29 for both groups, p < 0.0001). Numbers listed on top left corner of each panel indicate the mean and S.E.M. for each cell type. Inset shows Gaussian fits to data shown in histogram. HVCRA and HVCX cell body measurements derived from data published in Tschida and Mooney, 2012.

Supplementary Figure 2 Different HVCAv neurons show different activity patterns during singing in hearing-intact and deafened birds.

a) Field of GCaMP-labelled HVCAv neurons imaged through miniature microscope in a singing zebra finch in a single bird with hearing intact (left) and after deafening (right). Individual cells are indicated by colored outlines. Scale bars, 100 μm x 100 μm. b) Mean singing-related activity traces of five putative HVCAv neurons identified by CNMF analysis with hearing intact (left, n = 24 songs) and after deafening (right, n = 25 songs). Black lines represent mean activity traces for each neuron and the shaded area denotes ± one standard error of the mean response. Vertical scale bars, 10 (left) and 5 (right) arbitrary activity units; horizontal scale bars = 1 s and apply to traces in b – d; song motif onset is marked by vertical grey line. c) The summed activity from individual putative cell bodies shown in panel b). Vertical scale bars: 25 (left) and 20 (right) arbitrary activity units. d) Mean change in bulk fluorescence signals measured during singing from all birds with hearing intact (left, n = 4 birds) and after deafening (right, n = 2 birds) (same data as plotted in Figure 2c; scale bar 2% df/f). e) Cumulative probability distributions of mean decay time constants measured in singing zebra finches for GCaMP6s-expressing HVCAv neurons (blue, n = 16 ROIs) and a mixed population of GCaMP6s-expressing HVC neurons (red, n = 43 ROIs). The KS test p value is 0.9914 and the KS statistic is 0.1221. Raw imaging files to which the CNMF algorithm was applied are provided as Supplementary Movies 1 and 2.

Supplementary Figure 3 Effects of electrical stimulation on different HVC cell types.

a) Evoked EPSC in HVCAv cell after HVCRA stimulation at 80 (black trace) and 160 (grey trace) μA; scale bar 20 ms, 200 pA. b) Evoked IPSC in HVCAv cell after HVCRA stimulation at 80 uA; scale bar 20 ms, 300 pA. c) Evoked IPSC in HVCRA cell after HVCX stimulation at 80 μA; scale bar 20 ms, 50 pA. d) Evoked IPSC in HVCX cell after HVCX stimulation at 80 μA; scale bar 20 ms, 100 pA. e) Recording in HVCAv cell after electrical stimulation of LaM at 160 μA; scale bar 20 ms, 100 pA.

Supplementary Figure 4 Spectral derivatives illustrating learning outcome for all 5 (a–e) HVCAv lesioned birds.

Birds in panels a-c were tutored by the same adult male zebra finch in separate experiments. In panels a-d examples of copied syllable(s) are marked by a red line. The fifth zebra finch, in panel e, did not copy either of the two complex syllables in the tutors’ song. Birds and in panels a-b are the same birds illustrated in Figure 3 of the manuscript. Scale bar = 100ms (illustrated in bottom right of panel a).

Supplementary Figure 5 Cre-dependent Caspase 3 is efficacious and specific to targeted population.

a) Example of song from one bird before and after unilateral lesion of HVC from co-injection of Cre-dependent caspase 3 and Cre into HVC. b) Percent similarity to bird’s own song (self-similarity) before and after unilateral lesions of HVC for 2 birds after co-injection of Cre-dependent caspase 3 and Cre into HVC unilaterally. c) Average number of cells per 50 micron section of HVC in 2 birds in which HVCAv cells were unilaterally lesioned. HVCX cells, which were not targeted with the intersectional ablation strategy (blue), were comparable in number between unlesioned and lesioned hemispheres while HVCAv cells, which were targeted (red), showed a reduction by nearly half in lesioned hemispheres.

Supplementary Figure 6 Ablation of HVCAv in adult birds causes slight decreases in motif similarity and duration.

a) Experimental birds show a significant but slight decrease in percent similarity to bird’s own song (self-similarity) after HVCAv ablation (n = 12 HVCAv lesioned birds, paired two-sample t(11)=4.34, P = 0.001 comparing percent self-similarity before lesioning to percent self-similarity after lesioning in experimental birds.) b) Experimental birds show a significant decrease in motif duration after HVCAv ablation (n = 12 HVCAv lesioned birds, n = 12 HVCAv intact birds; two-sample t(22) = 2.49, P = 0.02 comparing percent change in motif duration from lesioned versus intact birds). c) Example sonograms of single syllables from two control birds before (top row) and after (bottom row) deafening. Scale bars, 50 ms. d) Example sonograms of single syllables from four experimental birds before (top row) and ten weeks after (bottom row) deafening. Scale bars, 50 ms.

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Roberts, T., Hisey, E., Tanaka, M. et al. Identification of a motor-to-auditory pathway important for vocal learning. Nat Neurosci 20, 978–986 (2017). https://doi.org/10.1038/nn.4563

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