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Unstable neurons underlie a stable learned behavior

Nature Neuroscience volume 19, pages 1665–1671 (2016)Cite this article

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Abstract

Motor skills can be maintained for decades, but the biological basis of this memory persistence remains largely unknown. The zebra finch, for example, sings a highly stereotyped song that is stable for years, but it is not known whether the precise neural patterns underlying song are stable or shift from day to day. Here we demonstrate that the population of projection neurons coding for song in the premotor nucleus, HVC, change from day to day. The most dramatic shifts occur over intervals of sleep. In contrast to the transient participation of excitatory neurons, ensemble measurements dominated by inhibition persist unchanged even after damage to downstream motor nerves. These observations offer a principle of motor stability: spatiotemporal patterns of inhibition can maintain a stable scaffold for motor dynamics while the population of principal neurons that directly drive behavior shift from one day to the next.

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Figure 1: Approach to measuring the stability of neural firing patterns underlying a highly stable behavior.
Figure 2: Activity patterns from multiunit ensembles and single interneurons are highly stable.
Figure 3: Drift in multiunit firing patterns is not accelerated by unilateral TS nerve transection.
Figure 4: A custom head-mounted fluorescence microscope adapted for use in singing birds.
Figure 5: Projection neurons drift over a 5-d imaging session.
Figure 6: Projection neuron activity preferentially changed over periods of sleep.

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Change history

  • 13 October 2016

    In the version of this article initially published online, all three colors in Figure 5c were labeled "Day 1." Green represents Day 2 and blue Day 3. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

The authors thank H. Eichenbaum and the Center for Neuroscience at BU for the loan of the Inscopix microscope. Special thanks to D.S. Kim and L. Looger for providing the GCaMP6 DNA and to the GENIE project at Janelia Farm Research Campus, Howard Hughes Medical Institute. This work was supported by a grant from CELEST, an NSF Science of Learning Center (SBE-0354378) and by grants from NINDS (R01-NS089679-01 and 1U01NS090454-01).

Author information

Author notes

  1. Jeffrey E Markowitz

    Present address: Present address: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.,

  2. William A Liberti and Jeffrey E Markowitz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Boston University, Boston, Massachusetts, USA

    William A Liberti III, Jeffrey E Markowitz, L Nathan Perkins, Daniel P Leman & Timothy J Gardner

  2. Graduate Program in Neuroscience, Boston University, Boston, Massachusetts, USA

    William A Liberti III & L Nathan Perkins

  3. Center for Regenerative Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

    Derek C Liberti & Darrell N Kotton

  4. The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA

    Derek C Liberti & Darrell N Kotton

  5. Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA

    Grigori Guitchounts

  6. Program in Neuroscience, Harvard University, Cambridge, Massachusetts, USA

    Grigori Guitchounts

  7. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Tarciso Velho & Carlos Lois

  8. Brain Institute, Federal University of Rio Grande de Norte, Natal, Brazil

    Tarciso Velho

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Contributions

W.A.L., J.E.M., G.G. and D.P.L. performed the experiment; J.E.M. and W.A.L. analyzed the data; L.N.P. provided software for the custom microscope; D.C.L., D.N.K., T.V. and C.L. provided the lentivirus; T.V. and C.L. discovered the cell-type-specificity of the viral vector. W.A.L., J.E.M. and T.J.G. designed the experiment and wrote the manuscript.

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Correspondence to Timothy J Gardner.

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Integrated supplementary information

Supplementary Figure 1 Ensemble measurements are stable over a month-long timescale.

(a) Phase difference of the 30 Hz LFP from 0-30 days (blue) and 31-511 days (red) (n=182 channels from n=21 implants). The mode for each distribution is shown as a dotted line. (b,c) Median RMS and firing rate (FR) during singing are shown for each bird (shown in separate colors) across the time-course of the experiment. Error bars indicate interquartile ranges.

Supplementary Figure 2 Unilateral tracheosyringeal (TS) nerve cut disrupts the acoustic details of song.

Example sonograms are shown for the 5 birds used in this experiment before (a) and after (b) TS nerve cut. The thin colored borders surrounding each sonogram match the colors used in Figure 3c-d.

Supplementary Figure 3 Histological verification of virus used for calcium imaging.

(a) Diagram of experimental strategy for calcium imaging. Virus expressing GCaMP6f was injected into HVC, and a retrograde dye (DiI) was injected into the downstream nucleus Area X. (b) Lentivirus with an RSV promoter produces dense infections of HVC projection neurons. Scale bar indicates 400 µm. Inset, closeup of HVC, bounded by the white dotted line. Scale bar indicates 39 µm. (c) Co-localization of GCaMP6f-expressing neurons in HVC and DiI backfill. Scale bar, 250 µm. (d) View of HVC through a chronically implanted GRIN lens. Scale bar, 250 µm. Experiment was repeated in n=2 of the 4 imaged birds.

Supplementary Figure 4 Schematic of custom camera and acquisition system.

(a) Signals from the camera can be wirelessly relayed with an off-the-shelf wireless transmitter (BOSCAM TX24019 or other). (b) Schematic of the data acquisition device used in this study. (c) To reduce cable weight and torque, our experiments made use of custom active commutators. These devices use the deflection of the magnetic field of a disk magnet located on a flex PCB cable to detect torque via a hall sensor (ALLEGRO MICROSYSTEMS A1301EUA-T). A feedback circuit mediated by a micro-controller (Arduino UNO) corrects the deflection by rotating a slip ring via a servo-driven gearbox with a 1:1 ratio.

Supplementary Figure 5 Summary of a 5-d longitudinal study from Figure 5a.

Overlay of 3 images, with each image representing the presence or absence of neural activity for a day’s worth of imaging, from an entire 5 day longitudinal study for one bird (GCaMP6s, commercial microscope).

Supplementary Figure 6 Cells change their participation in the motor sequence over periods of sleep

(a) Image overlays from morning and evening on a single day show minimal drift in cell participation. (b) Image overlays from evening to morning on a second day show that significant drift in cell participation occurred over the sleep interval.

Supplementary Figure 7 Examples of stable and unstable cells in the same imaging region.

(a) ROI masks for cells in region. (b) Maximum projection of a 191 x 203 µm subsection of the averaged, song-aligned calcium imaging movies, across 5 d. Arrows highlight two cells in this plane that either drop out (green, yellow) or in (blue) of the neural sequence across days. This region is a smaller section of the total imaging plane, shown in (c) and (d). (c) Three day maximum projection overlay from the data in Figure 5c. The maximum projection image is divided by a smoothed version of the same image (100 pixel disk filter) to normalize across bright and dim ROIs. (d) The last three days of the five day longitudinal study, using the same normalization as in (c).

Supplementary Figure 8 Examples of amplitude traces from multipeaked traces.

(a) Single frame stills from the trial-averaged, song-aligned calcium imaging movies the same animal shown in Supplementary Figure 7, across all 5 days of the longitudinal study. Columns are days, and rows are frame times relative to the start of song. (b) Example of three cells with stable timing and amplitude. Five traces represent five days (ordered top to bottom from day 1 to day 5.). (c) Traces of three cells that are unstable across days, with triangles indicating calcium peak times. Dashed lines indicate times corresponding to each frame from (a).

Supplementary Figure 9 Song shifts over periods of sleep in the adult zebra finch.

(a) Time-frequency probability densities, or spectral density images (SDIs, see Online Methods) were created for the first half and second half of song trials from two consecutive days (“day” is the first half, “night” the second) and overlaid. Top, day and night from the first day (Day 1 and Night 1, respectively), with Day 1 assigned to the blue channel and Night 1 to the red. Middle, Day 2 is assigned to the blue channel, with Night 1 again assigned to red. Bottom, the pixel correlation (SDI corr) between the red and blue channels is shown for each image, blue for Night 1-Day 1 and red for Night 1-Day 2. Increased scatter is found over intervals of sleep. (b) Scatter plots of the pixel values for each spectral density image. (c) Blow-ups of the regions highlighted in (a). Experiment repeated in n=10 birds.

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Liberti, W., Markowitz, J., Perkins, L. et al. Unstable neurons underlie a stable learned behavior. Nat Neurosci 19, 1665–1671 (2016). https://doi.org/10.1038/nn.4405

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