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Secondary auditory cortex mediates a sensorimotor mechanism for action timing

Nature Neuroscience volume 25, pages 330–344 (2022)Cite this article

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Abstract

The ability to accurately determine when to perform an action is a fundamental brain function and vital to adaptive behavior. The behavioral mechanism and neural circuit for action timing, however, remain largely unknown. Using a new, self-paced action timing task in mice, we found that deprivation of auditory, but not somatosensory or visual input, disrupts learned action timing. The hearing effect was dependent on the auditory feedback derived from the animal’s own actions, rather than passive environmental cues. Neuronal activity in the secondary auditory cortex was found to be both correlated with and necessary for the proper execution of learned action timing. Closed-loop, action-dependent optogenetic stimulation of the specific task-related neuronal population within the secondary auditory cortex rescued the key features of learned action timing under auditory deprivation. These results unveil a previously underappreciated sensorimotor mechanism in which the secondary auditory cortex transduces self-generated audiomotor feedback to control action timing.

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Fig. 1: Self-paced fixed-interval timing task in mice.
Fig. 2: Auditory deprivation acutely disrupts self-paced fixed-interval performance.
Fig. 3: Learning- and performance-dependent dorsal secondary auditory cortex cFos activation during self-paced fixed-interval task.
Fig. 4: Lever-pressing-related neuronal activity in dorsal secondary auditory cortex during the performance of self-paced fixed-interval task in trained mice.
Fig. 5: Dorsal secondary auditory cortex activation is necessary in providing the sensorimotor feedback for action timing.
Fig. 6: Press-dependent dorsal secondary auditory cortex activation is sufficient in providing the sensorimotor feedback for action timing.
Fig. 7: A computational model of action timing based on the integration of actions regulated by sensorimotor feedback.
Fig. 8: Dorsal secondary auditory cortex provides sensorimotor feedback during action timing via layer V active populations.

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Data availability

All data are available upon reasonable request from the corresponding author.

Code availability

All code is available upon request.

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Acknowledgements

The authors thank H. Bito for the ESARE viral construct, B. Sabatini for the DO-ChR2 viral construct, and M. Goulding, C. Kintner and J. Thomas for helpful discussion. This study was supported by grants from the National Institutes of Health under award numbers R01NS083815 and R01AG047669 to X.J., and EY022577 to E.M.C. and the McKnight Memory and Cognitive Disorders Award to X.J.

Author information

Authors and Affiliations

  1. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA

    Jonathan R. Cook, Hao Li, Bella Nguyen, Hsiang-Hsuan Huang, Payaam Mahdavian, Patrick Strassmann, Max Engelhardt & Xin Jin

  2. Neurosciences Graduate Program, University of California, San Diego, La Jolla, CA, USA

    Jonathan R. Cook, Hsiang-Hsuan Huang, Megan A. Kirchgessner & Patrick Strassmann

  3. Champalimaud Centre for the Unknown, Champalimaud Research, Champalimaud Foundation, Lisbon, Portugal

    Jonathan R. Cook

  4. Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, La Jolla, CA, USA

    Megan A. Kirchgessner & Edward M. Callaway

  5. Skirball Institute for Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    Megan A. Kirchgessner

  6. Center for Motor Control and Disease, Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai, China

    Xin Jin

  7. NYU–ECNU Institute of Brain and Cognitive Science, New York University Shanghai, Shanghai, China

    Xin Jin

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Contributions

X.J. conceived the behavior task. X.J. and J.R.C. conceived the sensorimotor hypothesis and designed the experiments. J.R.C., B.N. and P.M. conducted the behavioral experiments. J.R.C. and B.N. performed surgeries, histology and immunohistochemistry. J.R.C., P.M. and M.E. performed cell counting. J.R.C. and P.S. analyzed the behavioral data. H.L. and H.-H.H. performed the in vivo electrophysiological experiments and analyses in freely behaving mice during the performance of the SFI task. M.A.K. and E.M.C. performed the electrophysiological experiments and analyses in head-fixed FosTRAP mice. H.-H.H. performed the optogenetic experiments in Vgat-Ai32 mice. X.J. built the computational model and wrote the scripts. J.R.C. and X.J. wrote the manuscript.

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Correspondence to Xin Jin.

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Extended data

Extended Data Fig. 1 Self-initiated learning and performance of SFI peak timing is characterized notably by half peak PETH fall time, which reflects individual step-like lever press responding, related to Figs. 1 and 2.

a, Correlation analysis of average response rate (n = 10) at 30 s versus 70 s based on PETH for omission trials across training. b, The Pearson correlation coefficient can be calculated for the average response rate at each second over the omission trial window versus the average response rate at 30 s across learning showing autocorrelation around 30 s and a cross-temporal period of correlation around 70 s. The arrow denotes the Pearson correlation coefficient for the correlation in (a). c-d, Trial-by-trial analysis for the number of presses from 20–30 s and 60–70 s confirms the relationship between response rate changes at 30 s and 70 s from PETHs in (a). c, The response raster of an exemplar for the 5th trial of training days 1, 4, 7 and 21 shows that as the number of responses increase around 30 s, the number of responses around 70 s decreases. d, The numbers of responses over 20–30 s for the same exemplar in (c) negatively correlates with the number of responses over 60–70 s across learning for the 5th trial (top). The average number of presses for all animals (n = 10) for the 5th trial over 20–30 s negatively correlates with the average number of presses over 60–70 s (bottom). e-g, Response rasters (top), response rate PETH (middle), and percent maximum response rate PETH (bottom) of exemplars for rewarded (left) and omission trials (right) performing regular SFI (70% rewarded trials) on day 21 after being trained 20 days on either SFI with 70% (e) or 100% (f) rewarded trials. g-i, The latency to initiate post-lever extension does not change across training days (g), and does not show a relationship to half peak fall time (h) or peak time (i) at day 21 of training (n = 10). j, Alignment to the start and stop of pressing bouts reveals that peak timing is an artifact of averaging many trials. Bouts of pressing for an individual animal can be defined based on the trial-by-trial press rate to establish the start and stop (left, top, red hash marks) of pressing sequences that give rise to the overall PETH, which can be used to define the PETH peak time and half peak rise and fall time (left, bottom). Alignment of pressing bouts to the start (middle, top) and stop (right, top) reveals that PETHs resemble step functions (middle and right, bottom), indicating that trial-by-trial behavior is in fact characterized by low rates of responding early on, followed by an abrupt switch to a constant high rate, and then an abrupt return to no responding. k, Correlating PETH half peak fall time versus mean trial-by-trial stop time at day 21 of training reveals that the half peak fall time metric can serve as an accurate measure of overall individual trial stop times. l, Same as (k) but for PETH half peak rise time versus trial-by-trial start time. m-n, The PETH half peak rise time changes minimally across 21 days of training (m, main effect of treatment F(4, 36) = 11.54, P < 0.0001; day 1 vs. day 21, P< 0.0001), compared to half peak fall time (see Fig. 1), and shows nonsignificant changes with ear sealing (n). o-p, Analysis of interpress interval times between individual trial start and stop pressing times. The histogram of all interpress interval times between the start and stop times for all trials for the exemplar shown in (j) follows a Poisson-like distribution. (p) Autocorrelation function coefficients calculated for the sequence of interpress intervals across the second (top) and 10th (bottom) trials for increasing lag times, again for the exemplar shown in (j). Grey, non-labeled points denote intermediate training days for learning correlation plots. Error bars denote s.e.m. For correlation plots analyzing latency to initiate post-lever extension, and trial-by-trial mean stop time, grey points denote individual animals. Grey shading for all correlation plots denote 95% confidence interval for regression and PCC denotes Pearson correlation coefficient. **** P < 0.0001; NS, not significant. Blue dotted lines for autocorrelation functions denote rejection region bands for testing individual autocorrelations.

Extended Data Fig. 2 SFI ear seal learning, related to main Fig. 2.

a, Behavior of an example mouse for probe trials at day 1, 4, 7 and 21 of SFI ear seal training. b, Average training PETHs for ears open (left, n = 10) and ear sealed (right, n = 8) for response rate (top) and percent maximum response rate (bottom). c-e, The presses per minute at 30 s (c, effect of interaction F(4, 64) = 4.55, P = 0.0027; ears open vs. sealed: day 7, day 14, and day 21, P = 0.0009, P = 0.0008, and P = 0.0004, respectively), peak time (d), and half peak fall time (e, effect of interaction F(4, 64) = 3.43, P = 0.0133; ears open vs. sealed: day 4 and day 7, P < 0.0001 and P = 0.0139, respectively) across training days for ears open (n = 10) and sealed (n = 8). Learning data were analyzed using two-way ANOVA followed by Sidak post hoc comparisons. Values for performance metrics are means. Shading for average PETHs and error bars denote s.e.m. **** P < 0.0001, *** P < 0.001, * P < 0.05; NS, not significant.

Extended Data Fig. 3 Auditory deprivation effects on SFI performance are independent of any cues related to reward delivery, related to main Fig. 2.

a-e, Sweetened, condensed milk (n = 5) can be used as a reward for SFI training. a, Response rasters (top), response rate PETH (middle) and percent maximum response rate PETH (bottom) of an exemplar for omission trials performing SFI under auditory deprivation (ears sealed) between flanking control sessions (pre-control and post-control) using sweetened, condensed milk as a reward. SFI performance with milk reward can be measured by the response rate at 10 s (pre-control middle) and the half peak fall time (pre-control bottom) for omission trials. Ear sealing has no effect on the rewarded head entry time (b), or how often animals check for the milk reward by making a head entry (c), indicating auditory deprivation does not have an effect on a sensory cue related to reward availability. d, Auditory deprivation experiments on animals trained using the milk reward showed similar effects on response rate (left, main effect of treatment F(2,8) = 9.209, P = 0.0084; ears sealed versus pre-/post-control, P = 0.0115 and P = 0.0191, respectively) and half peak fall time (right, main effect of treatment F(2,8) = 9.414, P = 0.0079; ears sealed versus pre-/post-control, P = 0.0107 and P = 0.0183, respectively) as with the pellet reward. e, Average PETHs for response rate (top) and percent maximum response rate (bottom) for performance with sweetened, condensed milk reward on pre-control session and ear seal session. f-h, Separate groups of animals underwent extinction through exposure to continuous omission trials with ears open (n = 8) or sealed (n = 10) also demonstrating that the auditory deprivation effects on SFI performance were independent of a reward-related cue. f, Under auditory deprivation during extinction, response rate at 30 s decreased (left, two-tailed, unpaired t-test, t = 5.076, P = 0.0001), half peak fall time increased (right, two-tailed, unpaired t-test, t = 3.347, P = 0.0041), and no significant change was observed in the peak time. g, Response rasters (top), response rate PETH (middle), and percent maximum response rate PETH (bottom) of exemplars for omission trials performing extinction post-21 days of training on SFI with ears open (left) and sealed (right). h, Average PETHs for response rate (top) and percent maximum response rate (bottom) for ears sealed and open groups. SFI milk data were analyzed using repeated-measures one-way ANOVA followed by Tukey post hoc comparisons. Extinction results were analyzed using unpaired t-tests. Values for performance metrics are means, and error bars and shading for average PETHs denote s.e.m. ***P < 0.001; ** P< 0.01; *P< 0.05; NS, not significant.

Extended Data Fig. 4 Effects of unilateral auditory deprivation on cFos expression in auditory structures and striatum, related to main Fig. 3.

a, cFos expression in AUDd (left), ipsilateral to the sealed ear (middle), and ipsilateral to the open ear (right) of a unilateral ear sealed animal. (b-d), same as (a), but for the medial geniculate (b), inferior colliculus (c), and striatum (d). Scale bars for immunohistochemical images denote 200 μm for auditory structures, and 1 mm for striatum. ‘D’ and ‘L’ denote dorsal and lateral, respectively.

Extended Data Fig. 5 Effects of unilateral auditory deprivation on the visual system activation, and validation of FosTRAP labeling system in AUDd, related to main Figs. 3 and 4.

a-b, Random unilateral ear sealing does not disrupt cFos expression across hemispheres in VISp. a, cFos expression across hemispheres in VISp was quantified in the same group of animals that underwent random unilateral ear sealing while performing SFI and then sacrificed. b, cFos immunohistochemistry for exemplar showing VISp cortical region for an animal sacrificed on day 21 of SFI training with one ear sealed (left) and one ear open (middle). Scale bars denote 50 μm. Comparison of percent activation according to cFos counts across hemispheres for VISp cortical region (right) ipsilateral versus contralateral to the sealed ear of animals sacrificed upon completion of SFI at day 21 (n = 5) of training. c-g, FosTRAP expression recapitulates cFos protein expression pattern in AUDd. c, FosTRAP animals were trained for 21 days on SFI and split into two groups. One group (top)(n = 4) was induced with 4-OHT after being injected with a Cre-dependent AAV expressing GFP in AUDd and eventually sacrificed immediately after performing SFI again with ears open. The other group (bottom)(n = 4) performed the no lever version of the task also with ears open and immediately sacrificed following session completion. d, Percent distribution across the cortical layers in AUDd of cFos protein and cFosCreERT2 (as visualized via Cre-dependent GFP expression) in FosTRAP animals that were induced (green) while performing SFI with their ears open and later in another SFI session immediately sacrificed upon completion (black), again with ears open. These distributions were compared to the cFos protein percent distribution of FosTRAP animals that were sacrificed upon session completion in the no lever context with their ears open (red)(effect of interaction F(8,45) = 2.335, P = 0.0343; no lever layer V cFos protein vs. SFI layer V cFosCreERT2/cFos protein, P = 0.0172 and P = 0.0496, respectively) with ears open (n = 4). e-f, AUDd cortical layer expression pattern of endogenous cFos protein for FosTRAP animals sacrificed immediately after performing SFI (e) and being in the no lever context (f) with their ears open. g, FosTRAP cFosCreERT2 expression as visualized via Cre-dependent GFP expression in AUDd of an animal induced with its ears open while performing SFI. Scale bars denote 100 μm. h, Fiber placement in AUDd of FosTRAP animals injected with an AAV expressing Cre-dependent ChR2-EYFP and induced with its ears open while performing SFI. Scale bar denotes 500 μm. For VISp cFos expression analysis, data were analyzed using a paired t-test. Bars denote mean percentage across hemispheres. For FosTRAP validation and AUDd laminar analysis, data were analyzed using two-way ANOVA followed by Sidak post hoc comparisons. Values are mean percentages across layers. Error bars denote s.e.m. For all immunohistochemistry images, ‘D’ and ‘L’ denote dorsal and lateral, respectively. *P < 0.05; NS, not significant.

Extended Data Fig. 6 Firing properties of AUDd neurons with ears open versus sealed and photoactivation of AUDd FosTRAPed populations, related to main Figs. 4 and 6.

a, Baseline firing rates of AUDd neurons with lever press responses exhibit no significant difference between ears open and sealed (n = 83). b, Raw firing rate (that is not normalized to baseline firing rate) between ears open and sealed for neurons with activated responses (left, n = 11) upon lever pressing significantly decreased their peak firing rate (open: 17.0 ± 4.7 Hz; sealed: 14.2 ± 5.0 Hz; two-tailed, paired t-test, t = 2.480, P = 0.0325), while neurons with inhibited responses (right, n = 5) showed no significant change with ear sealing. c, Latency of peak/dip responses upon lever pressing between ears open and sealed for activated (left, n = 11) and inhibited (right, n = 5) neurons showed no significant difference. d, Single-units were recorded extracellularly from AUDd in FosTRAP animals (n = 2, 2 recording sessions) in which ChR2-EYFP was expressed in a Cre-ON dependent manner following 4-OHT induction. e, Raster plot (left) and PSTH (right) of an example photo-tagged unit (significant response within 10 ms of light stimulus onset; P < 0.01, Stimulus-Associated spike Latency Test (SALT)). f, Light modulation index (difference in light-evoked and baseline firing rate divided by their sum) of all single-units recorded at different cortical depths. Filled dots indicate putatively photo-tagged units (P < 0.01, SALT). Bars indicate the mean light modulation index in 100 μm bins (unfilled: all units, filled: photo-tagged units). Pie chart: percent of single-units that are putatively photo-tagged (P < 0.01, SALT). g, Single-units were recorded extracellularly from AUDd in FosTRAP animals (n = 2, 3 recording sessions) in which ChR2-mcherry was expressed in a Cre-OFF dependent manner following 4-OHT induction. h-i, Same as for (e-f) but for Cre-OFF population. j-l, Correlation analysis of change in the press rate at 30 s and change in half peak fall time between stimulation and no stimulation trials (Δ = stimulation - no stimulation) for the three experimental conditions tested with FosTRAP: ears open/Cre-ON (j), ears sealed/Cre-ON (k), and ears open/Cre-OFF (l). Firing indicies are means, and error is s.e.m. Differences in firing index were analyzed using paired t-tests. *P < 0.05; NS, not significant. Grey shading for correlation plots denotes 95% confidence interval for regression. PCC denotes Pearson correlation coefficient.

Extended Data Fig. 7 FosTRAPed populations, and assessment of muscimol spread, related to main Fig. 4.

a, Exemplar histology from a FosTRAP animal showing Cre-dependent expression of EYFP is primarily restricted to AUDd. Optrode placement is overlayed showing conical light spread from the fiber tip. b, Cell density of Cre-ON cells in the three auditory cortical fields (top), and percentage within each field (bottom). The highest density (main effect of treatment F(2, 9) = 4.305, P = 0.0488, AUDd vs. AUDv, P = 0.0439) and percentage (main effect of treatment F(2, 9) = 17.75, P = 0.0008, AUDd vs. AUDv, P = 0.0006) of cells are located within AUDd, with decreasing expression moving ventrally to AUDv (n = 4). c, Diagram depicting parameters used to calculate conical volume of illumination sufficient to induce spiking. Using a formula that takes into account scattering, absorption, and geometric loss of light through the brain, we calculated the total illuminated volume that is within the intensity threshold (≥1 mW/mm2) to activate ChR2 and elicit spikes. This volume, which is based on the 5 mW initial intensity at the fiber tip (~159 mW/mm2) makes up a cone with an angle of θ = 33° that stretches z = 0.87 mm downward. Using the trigonometric relationship between z and θ, the radius of the base of this cone, r = 0.061 mm3, can be calculated. Based on r and z, the volume of the cone can be determined. Using the neuronal density of the auditory cortex, and the percentage of Cre-ON and Cre-OFF cells from laminar recordings, we calculated ~1,100 cells/hemisphere were activated in the ears open/Cre-ON condition, and ~5,600 cells/hemisphere in the ears open/Cre-OFF condition. d, Exemplar histology of an animal implanted with a cannula in AUDd, and sacrificed following infusion of fast green, showing dye is largely restricted to AUDd. For FosTRAP cell density and regional percentage quantifications, data were analyzed using one-way ANOVA followed by Tukey post hoc comparisons, and bars are means. Scale bars for all immunohistochemical images denote 1 mm, and ‘D’ and ‘L’ denote dorsal and lateral, respectively. ***P < 0.001; *P < 0.05; NS, not significant.

Extended Data Fig. 8 Optogenetic, press-dependent perturbation of AUDd via stimulation of CAMKII + populations with ears open disrupts SFI timing performance, related to main Fig. 5.

a, Exemplar histology from a CAMKII-Cre animal showing Cre-dependent expression of ChR2-EYFP is primarily restricted to AUDd. b, Behavior of an example animal using closed-loop, press-triggered optical stimulation (5 mW, 100 ms per press) of CAMKII + populations in AUDd during SFI performance (blue line denotes stimulation and grey denotes no stimulation). c, Stimulation and no stimulation average PETHs (n = 8) for response rate (top) and percent maximum response rate (bottom) for press triggered optical stimulation of CAMKII + populations in AUDd with ears open. d, The optogenetic stimulation effects on response rates at 30 s (left, two-tailed, paired t-test, t = 4.295, P = 0.0036), peak time, and half peak fall times (right, two-tailed, paired t-test,t = 5.261, P = 0.0012) for press-dependent optical stimulation of CAMKII + populations in AUDd (n = 8) during SFI performance with ears open. Scale bars for immunohistochemical image denotes 400 μm, and ‘D’ and ‘L’ denote dorsal and lateral, respectively. Shading for average PETHs denotes SEM. **P < 0.01; NS, not significant.

Supplementary information

Reporting Summary

Supplementary Video 1

Behavioral example of a trained mouse during the probe trial of the SFI task.

Supplementary Video 2

Behavioral example of a FosTRAP mouse from the ears-open/Cre-ON group performing the SFI task under auditory deprivation during probe trials without (top) or with (bottom) optical stimulation.

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Cook, J.R., Li, H., Nguyen, B. et al. Secondary auditory cortex mediates a sensorimotor mechanism for action timing. Nat Neurosci 25, 330–344 (2022). https://doi.org/10.1038/s41593-022-01025-5

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