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Delayed motor learning in a 16p11.2 deletion mouse model of autism is rescued by locus coeruleus activation

Nature Neuroscience volume 24, pages 646–657 (2021)Cite this article

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Abstract

Children with autism spectrum disorder often exhibit delays in achieving motor developmental milestones such as crawling, walking and speech articulation. However, little is known about the neural mechanisms underlying motor-related deficits. Here, we reveal that mice with a syntenic deletion of the chromosome 16p11.2, a common copy number variation associated with autism spectrum disorder, also exhibit delayed motor learning without showing gross motor deficits. Using in vivo two-photon imaging in awake mice, we find that layer 2/3 excitatory neurons in the motor cortex of adult male 16p11.2-deletion mice show abnormally high activity during the initial phase of learning, and the process of learning-induced spine reorganization is prolonged. Pharmacogenetic activation of locus coeruleus noradrenergic neurons was sufficient to rescue the circuit deficits and the delayed motor learning in these mice. Our results unveil an unanticipated role of noradrenergic neuromodulation in improving the delayed motor learning in 16p11.2-deletion male mice.

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Fig. 1: Delayed motor learning in 16p11.2+/− mice.
Fig. 2: Abnormally high ensemble activity during the initial phase of motor learning in 16p11.2+/− mice.
Fig. 3: Delayed spine pruning in 16p11.2+/− mice.
Fig. 4: Fewer LC-NA neurons are activated in 16p11.2+/− mice during motor learning.
Fig. 5: Pharmacogenetic activation of LC-NA neurons in 16p11.2+/−::DBH-CreERT mice lowered the abnormally high ensemble activity and reversed the delayed spine pruning.
Fig. 6: Pharmacogenetic activation of LC-NA neurons in 16p11.2+/−::DBH-CreERT mice rescued the delayed motor learning.

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Data availability

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

Code availability

The codes used for analyzing the data in the current study are available from the corresponding author upon request.

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Acknowledgements

We thank B. Lacoste for sharing the 16p11.2+/− mice; B. Liu, J.C. Beique, M. Rousseaux, R. Naud and members of the Chen laboratory for comments and discussions; and B.H. Liu, H. Kato, T. Bui and M. Rousseaux for providing feedback on the manuscript. This work was supported by grants for S.X.C. from the Canada Research Chair (CRC) (grant no. 950-231274), the Canadian Institutes of Health Research (CIHR) (grant no. 153254), the Natural Sciences and Engineering Research Council of Canada (NSERC) (grant no. 05308), the Simons Foundation, the Scottish Rite Charitable Foundation, and the Brain & Behavior Research Foundation (NARSAD) (grant no. 27177).

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Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

    Xuming Yin, Nathaniel Jones, Jungwoo Yang, Nabil Asraoui, Marie-Eve Mathieu, Liwen Cai & Simon X. Chen

  2. Brain and Mind Research Institute, University of Ottawa, Ottawa, Ontario, Canada

    Simon X. Chen

  3. Center for Neural Dynamics, University of Ottawa, Ottawa, Ontario, Canada

    Simon X. Chen

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Contributions

X.Y. and S.X.C. conceived the project. All the experiments were performed by X.Y. with help from N.J., J.Y., N.A., M.-E.M. and L.C. N.J. collected the behavior data and performed the DeepLabCut analyses. J.W. collected and analyzed the data for the colocalization experiments of LC-NA neurons. N.A. collected and analyzed the data for ChAT-positive axons in motor cortex, and collected and analyzed the behavior data for the administration of muscimol and noradrenergic receptor antagonists. M.-E.M. collected and analyzed the data for NET-positive axons in motor cortex. L.C. helped with collecting the calcium imaging data and calcium imaging data analysis. All the rest of the data were collected and analyzed by X.Y. under supervision of S.X.C. X.Y., N.J. and S.X.C. wrote the manuscript.

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Correspondence to Simon X. Chen.

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Extended data

Extended Data Fig. 1 Mice showed a decrease in movement variation with motor learning in the rotating-disk task.

a, Schematic of the rotating-disk task recorded by a high speed and resolution camera from the top. b-c, DeepLabCut was used to unbiasedly track the position of head (white), neck (black), thorax (white), and sacrum (orange) across 12 sessions. Representative plots of all the corporeal coordinates from the neck and sacrum and the calculated angles from one RE of a mouse in session 1 (b) and session 12 (c). a.u., arbitrary unit. d, Mean relative variance of the angles decreased with motor learning (F(3,20) = 5.96, P = 0.004; 1-way ANOVA; Solid line, mean variance; shading, s.e.m.; n = 5 mice). e, Mean relative variance of the angles in ‘early’ (first 1/3 of the session) and ‘late’ (last 1/3 of the session) groups in session 1, 4, 8, and 12. Both ‘early’ and ‘late’ groups were comparable to each other throughout learning (n = 5 mice). One-tailed bootstrap test. Error bars indicate s.e.m.

Extended Data Fig. 2 WT and 16p11.2+/− mice showed similar performance on the head-fixed rotating-disk task in session 1.

a, Schematic of the rotating-disk task. b-c, Mean running attempts (b), distance travelled (c) in session 1. Both groups showed a comparable number of running attempts but had low total distance travelled (WT, n = 10 mice; 16p11.2+/−, n = 7 mice). One-tailed bootstrap test. d-e, Mean RE counts (d) and RE duration (e) in session 1. Both groups showed comparable performance in session 1 (WT, n = 10 mice; 16p11.2+/−, n = 7 mice). One-tailed bootstrap test. f, Mean probability distribution of sub-threshold velocities during SEs in session 1 (n = 7 mice per group). Both groups showed a comparable distribution of velocities. Error bars indicate s.e.m.

Extended Data Fig. 3 The elevated ensemble activity is RE-specific, which only occurs during the initial learning phase in 16p11.2+/− mice and is not related with the number of RE-active neurons or the ensemble activity during SEs.

a, Schematic of the two-photon Ca2+ imaging on the rotating-disk task. b, Representative of all the regions of interests (ROIs) drawn from one mouse in session 12. Each ROI represents a neuron. The mean ROIs analyzed in session 12 was 400 ± 48 neurons. Scale bar = 100 µm. c, Frequency of synchrony events from ‘pre-existing RE-active neurons’ in session 1-3 (same population as Fig. 2e) and followed throughout learning. ‘Pre-existing RE-active neurons’ in 16p11.2+/− mice initially showed more synchrony events, which desynchronized with training (F(3,20) = 3.27, P = 0.043, 1-way ANOVA). In contrast, ‘pre-existing RE-active neurons’ in WT mice had low synchrony and remained constant throughout learning (F(3,20) = 0.14, P = 0.933, 1-way ANOVA). One-tailed bootstrap test. d, Median activity index from ‘all RE-active neurons’ in session 1-3, 4-6, 7-9, and 10-12. Only ‘all RE-active neurons’ in 16p11.2+/− mice initially (session 1–3) showed higher activity than WT mice. One-tailed bootstrap test. e, Frequency of synchrony events from ‘all RE-active neurons’ in session 1–3, 4–6, 7–9, and 10–12 (same population as d). Only ‘all RE-active neurons’ in 16p11.2+/− mice initially (session 1–3) showed elevated synchrony events compared to WT mice. One-tailed bootstrap test. f, The number of ‘all RE-active neurons’ across training was similar between WT and 16p11.2+/− mice. One-tailed bootstrap test. g, The number of ‘newly-emerged RE-active neurons’ across training was similar between WT and 16p11.2+/− mice. One-tailed bootstrap test. h, Mean ΔF/F0 during SEs from all RE-active neurons in session 1–3. One-tailed bootstrap test. i, Median activity index of RE-active neurons in session 1–3, calculated between sub-threshold events and stationary events during SEs. One-tailed bootstrap test. j, k, Median activity index (j) and synchrony events (k) from SE-active neurons in session 1–3. Both WT and 16p11.2+/− mice showed a similar level of activity and synchrony events. One-tailed bootstrap test. l, The number of ‘newly-emerged RE-active neurons’ during training (session 4–6) was not correlated with the improvement in task performance (session 4–7) (Pearson linear correlation coefficient, r = −0.304, P = 0.337). m, Schematic of the two-photon spine imaging on the rotating-disk task. n, Low magnification images for the L2/3 distal dendrites (left) and somatic (right) regions of L2/3 excitatory neurons. Scale bar = 50 µm. **P < 0.01, ***P < 0.001 Error bars indicate s.e.m.

Extended Data Fig. 4 16p11.2+/− mice retain the memory of the head-fixed rotating-disk task.

a, Left: schematic of the head-fixed rotating-disk task. Right: Experimental timeline. Orange lines indicate the spine imaging sessions during retraining. P, postnatal day. b–d, Behavioral performances in the last session of training (Training_S12), the first session of retraining (Retraining_S1), and the last session of retraining (Retraining_S7). Both groups retained the motor memory and had comparable mean total running distance travelled (b), RE counts (c), and RE duration (d) between all 3 sessions (WT, n = 6 mice; 16p11.2+/−, n = 5 mice). One-tailed bootstrap test with Bonferroni correction. e-g, Mean spine density (e), pre-existing spine elimination (f), and spine dynamics between every 3 sessions during retraining (g) showed no significant changes (WT, n = 3 mice, 136 spines; 16p11.2+/−, n = 3 mice, 154 spines). 2-way ANOVA. Error bars indicate s.e.m.

Extended Data Fig. 5 Number of VTA-DA and BF-ACh activated neurons are not affected in 16p11.2+/− mice.

a, b, Representative coronal sections with immunostaining from 1-day rotarod trained mice. a, Left: a coronal section stained for c-Fos (green) and tyrosine hydroxylase (TH, red) (~3.3 mm posterior from the bregma). VTA and hippocampus are emphasized by dotted boxes. Scale bar = 1,000 µm. Middle: example images of c-Fos+ cells, TH+ cells, and co-localized cells in the VTA and hippocampus in WT and 16p11.2+/− mice. Scale bar = 100 µm. Right: mean percentage of c-Fos+ cells among the dopaminergic neurons in VTA. A comparable level of c-Fos+-TH+ neurons was found between the two groups (n = 6 mice). One-tailed bootstrap test. b, Left: a coronal section stained for c-Fos (green) and choline acetyltransferase (ChAT, red) (~0.9 mm posterior from the bregma). Basal forebrain and hypothalamus are emphasized by dotted boxes; scale bar = 1,000 µm. Middle: example images of c-Fos+ cells, ChAT+ cells, and co-localized cells in the basal forebrain and hypothalamus in WT and 16p11.2+/− mice. Scale bar = 100 µm. Right: mean percentage of c-Fos+ cells among the cholinergic neurons in basal forebrain. A comparable level of c-Fos+-ChAT+ neurons was found between the two groups (n = 6 mice). One-tailed bootstrap test. c, Mean DAPI density in the L2/3 of the motor cortex. No significant difference was found between the two groups (n = 5 mice). One-tailed bootstrap test. d, ChAT staining to measure cholinergic axonal innervation in the L2/3 of the motor cortex. Mean innervation density showed there was no significant difference between the two groups (n = 3 mice). One-tailed bootstrap test. e, NET staining to measure noradrenergic axonal innervation in the L2/3 of the somatosensory cortex. Mean innervation density showed there was no significant difference between the two groups (n = 6 mice). One-tailed bootstrap test. Error bars indicate s.e.m.

Extended Data Fig. 6 Pharmacogenetic activation restored the number of activated LC-NA neurons in 16p11.2+/− mice.

a, Left: schematic of the brain section used to stain for activated c-Fos and NA neurons in the LC. LC is emphasized by the orange shadow. Right: example images of hM3Dq-mCherry (far left), TH+ (center left), c-Fos+ (center right), and co-localized (far right) in the LC-NA neurons from 16p11.2+/−::DBH-CreERT+/− mice expressing hM3Dq-mCherry. Scale bar = 50 µm. Displayed images are representative of experiments repeated in five mice with similar results obtained. b, Mean percentage of LC-NA neurons expressing hM3Dq-mCherry among the total LC-NA neurons. c, Mean percentage of NA neurons that are c-Fos+ in WT mice (n = 6 mice), 16p11.2+/− mice (n = 5 mice), and hM3Dq group (n = 5 mice) after one session of the rotating-disk training. One-tailed bootstrap test with Bonferroni correction. d, Mean total number of LC-NA neurons in WT mice (n = 6 mice) and 16p11.2+/− mice (n = 6 mice). One-tailed bootstrap test. e, Schematic of the head-fixed rotating-disk task. f–h, Mean total distance travelled (f), RE counts (g), and RE duration (h) in session 1 of the four experimental groups in the DREADDs experiments. All four groups showed comparable performance. One-tailed bootstrap test. **P < 0.01 Error bars indicate s.e.m.

Extended Data Fig. 7 Proposed model for circuit dysfunctions during delayed motor learning in 16p11.2+/− mice.

Left: motor learning induces circuit reorganizations in the motor cortex through a multi-step process: In WT mice, during the initial phase of learning, RE neurons explore learning-related activity patterns, and nascent spines are formed to create structural substrates for the new motor memory (1). As learning progresses, NA is being released and ‘task-related’ RE-active neurons refine learning-related activity patterns (2). The refinement process to generate learned movement leads to the elimination of unnecessary spines to achieve proper circuit reorganization (3). Right: in the 16p11.2+/− mice, the process of circuit reorganization is delayed. The low levels of NA in the motor cortex of 16p11.2+/− mice could alter the baseline excitability of neurons. Hence, during an RE in the initial phase of learning, ‘task-related’ RE-active neurons become highly active, which leads to elevated ensemble synchrony but learning-induced spine formation is not affected (1). As learning progresses, NA is being released, and neuronal excitability reverts to baseline levels. As the ensemble begins to desynchronize (2), it allows ‘task-related’ RE-active neurons to refine learning-related activity patterns (3). However, the extra step of lowering neuronal activity and desynchronization delays the circuit’s ability to refine activity patterns and eliminate unnecessary spines to achieve proper circuit reorganization (4); thereby causing the delayed motor learning.

Supplementary information

Supplementary Information

Supplementary Table 1. Summary of the number of mice, spines, branch number, branch length and density in all experimental conditions.

Reporting Summary

Supplementary Video 1

WT mouse underwent head-fixed rotating-disk training in session 1. The mouse showed multiple movement attempts but was not able to smoothly control the rotation of the bi-directional disk under head-fixation.

Supplementary Video 2

WT mouse underwent head-fixed rotating-disk training in session 12. The mouse learned to modify its gait strategy to reduce movement variability and facilitate continuous running on the disk.

Supplementary Video 3

In vivo two-photon imaging of LC-NA axons expressing hM3Dq-mCherry in the motor cortex 4 weeks after the viral injection. The stack of images was taken at 512 × 512 pixels covering 503 × 429 µm2 with a 1-µm z-axis step size (100–208 µm from the pial surface) between planes. The mCherry-expressing axons were abundant and covered almost the entire imaging plane.

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Yin, X., Jones, N., Yang, J. et al. Delayed motor learning in a 16p11.2 deletion mouse model of autism is rescued by locus coeruleus activation. Nat Neurosci 24, 646–657 (2021). https://doi.org/10.1038/s41593-021-00815-7

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