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Astrocytic Ca2+ prevents synaptic depotentiation by limiting repetitive activity in dendrites during motor learning

Nature Neuroscience (2025)Cite this article

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Abstract

Astrocytic Ca2+ activity regulates activity-dependent synaptic plasticity, but its role in learning-related synaptic changes in the living brain remains unclear. We found that motor training induced synaptic potentiation on apical dendrites of layer 5 pyramidal neurons, as well as astrocytic Ca2+ rises in the mouse motor cortex. Reducing astrocytic Ca2+ led to synaptic depotentiation during motor training and subsequent impairment in performance improvement. Notably, synaptic depotentiation occurred on a fraction of dendrites with repetitive dendritic Ca2+ activity. On those dendrites, dendritic spines that were active before dendritic Ca2+ activity underwent CaMKII-dependent size reduction. In addition, the activation of adenosine receptors prevented repetitive dendritic Ca2+ activity and synaptic depotentiation caused by the reduction of astrocytic Ca2+, suggesting the involvement of ATP released from astrocytes and adenosine signaling in the processes. Together, these findings reveal the function of astrocytic Ca2+ in preventing synaptic depotentiation by limiting repetitive dendritic activity during learning.

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Fig. 1: Motor training induces transient elevation of Ca2+ in somas and processes of astrocytes.
Fig. 2: Motor training-induced astrocytic Ca2+ transient is dependent on neuronal activity and Ca2+ release from the ER.
Fig. 3: Astrocytic Ca2+ activity is important for motor training-induced synaptic potentiation.
Fig. 4: Astrocytes promote dendritic spine plasticity and performance improvement after learning.
Fig. 5: Reduction of astrocytic Ca2+ increases repetitive dendritic Ca2+ activity after the first 10–30 s of motor training.
Fig. 6: Synaptic depotentiation occurs on dendrites with repetitive Ca2+ activity.
Fig. 7: Astrocytes regulate spike-timing-dependent synaptic depotentiation during motor learning.
Fig. 8: Adenosine signaling is involved in astrocyte-dependent synaptic plasticity.

Data availability

All data supporting the findings of this study are provided within the paper and its Supplementary Information. Source data are provided with this paper.

Code availability

MATLAB codes used to detect astrocytic fine processes in this study are available in Code Ocean (https://doi.org/10.24433/CO.6561192.v1)64.

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Acknowledgements

We thank E. Sigurdsson, and all the members of the Guang, Gan and Chao laboratories for valuable comments on the paper. We thank S. Teng, A. Mar, M. Cui, Q. Hu, Y. Morizawa, X. Wang, Z. Cheng and W. Li for advice and support. This study received institutional support from NYU Medical Center and funding from the Alzheimer’s Association (24AARF-1246764, to B.L.), STI2030-Major Projects (2022ZD0211900), Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions (2024SHIBS0004) and Major Program of Shenzhen Bay Laboratory (S241101005).

Author information

Authors and Affiliations

  1. Department of Neuroscience, Institute for Translational Neuroscience, New York University Grossman School of Medicine, New York, NY, USA

    Baoling Lai, Alejandro Martín-Ávila & Moses V Chao

  2. Shenzhen Bay Laboratory, Shenzhen, China

    Deliang Yuan, Zhiwei Xu, Feilong Zhang & Wen-Biao Gan

  3. Peking University Shenzhen Graduate School, Shenzhen, China

    Deliang Yuan, Zhiwei Xu, Feilong Zhang, Kunfu Ouyang & Wen-Biao Gan

  4. Faculty of Psychology, Beijing Normal University, Beijing, China

    Ming Li

  5. Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA

    Xufeng Chen

  6. Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA

    Kai Chen & Guang Yang

  7. Lingang Laboratory, Shanghai, China

    Wen-Biao Gan

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  1. Baoling Lai
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  2. Deliang Yuan
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  3. Zhiwei Xu
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  11. Moses V Chao
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Contributions

Initiated and conceived the study: W.-B.G. and B.L. Experimental design: B.L., W.-B.G. and Z.X. Astrocytic Ca2+ imaging: B.L., Z.X., A.M.-A. and D.Y. Astrocytic ER Ca2+ imaging: B.L., K.C., X.C. and G.Y. Treadmill training/behavioral analysis: B.L. and D.Y. Longitudinal structural imaging: B.L., F.Z. and Z.X. Dendrite/dendritic spine Ca2+ imaging: B.L., Z.X., F.Z. and A.M.-A. Adenosine imaging: B.L., D.Y. and X.C. Data analysis and visualization: B.L., M.L., X.C. and W.-B.G. MATLAB coding: M.L. and B.L. Mouse strain maintenance and genotyping: K.O., B.L. and D.Y. Project supervision: W.-B.G., M.C. and B.L. Paper writing and preparation: B.L. and W.-B.G.

Corresponding authors

Correspondence to Baoling Lai or Wen-Biao Gan.

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Extended data

Extended Data Fig. 1 Astrocytes show increases of global and local Ca2+ during motor training in transgenic mice expressing GCaMP5 and tdTomato.

(a) Images of layer 1 astrocytes expressing both GCaMP5 and td-Tomato (GLAST-CreER;PC::G5-tdT) during pre-run and running conditions. Nearly all the astrocytes with td-Tomato labeling showed elevated GCaMP5 during motor running at 11 s (n = 61 cells, 4 mice). Scale bar, 20 μm. (b, c) Heatmaps (top) and amplitude (bottom) of Ca2+ transients in somas (b) and processes (c) of astrocytes during the periods of pre-run and 2-min treadmill running (n = 4 mice). (d) The average amplitude of astrocytic Ca2+ transients in the somas (n = 61) and processes (n = 1412) during pre-run and different periods of treadmill running. Two sided paired t test. ***P < 0.001. (e) Simultaneous imaging of neuronal and astrocytic activity in neuropils expressing GCaMP6s and astrocytes expressing both GCaMP5 and Td-tomato (GLAST-CreER;PC::G5-tdT), and fluorescence traces of different astrocytes and neuropils corresponding to the regions of interests (ROI) shown (up), during pre-run and running periods. Scale bar, 30 μm. (f) Neuropil activity (in black, n = 5 Thy1.2-GCaMP6s mice) increased immediately after running and its peak proceeded the peak of astrocytic Ca2+ (in green, n = 4 control mice with GCaMP6f indicators expressed in astrocytes); Note that neuropil activity decreased when astrocytic Ca2+ transients peaked. Both of neuropil and astrocytic activities were reduced in the prolonged period (60-110 s) of running trial. Two sided paired t test. P = 0.0083, P = 0.0054. (g) The dendritic Ca2+ activity during 0-30 s and 60-110 s of running (n = 244). Two sided paired t test. P < 0.001. Data are mean ± s.e.m. **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 2 Adrenergic α1 signaling plays an important role in the generation of astrocytic Ca2+ during motor training.

(a) Schematic of the experimental design to determine neuronal and astrocytic Ca2+ activity during running with TTX followed by PE treatment. (b) Images representing 7 independent experiments of running-induced neuronal activity (Thy1.2-GCaMP6s mice) before (ACSF) and after local application of TTX followed by PE. Scale bar, 20 μm. (c) The average amplitude of running-induced dendritic Ca2+ changes after treatment with ACSF, TTX, and PE following TTX (n = 94). One-way ANOVA. Boxes show the first quartile, median and third quartile of the stack data. Whiskers show minima and maxima without outliers. (d) Images representing 3 independent experiments of astrocytic Ca2+ activity with TTX (running), PE following TTX (resting), and PE-only administration (resting, n = 4 mice). Scale bar, 20 μm. (e) The average amplitude of astrocytic Ca2+ changes in the somas (n = 213, 217, 178 respectively) and processes (n = 1482, 1482, 984 respectively) with TTX (running), PE following TTX (resting) and PE-only (resting). Two sided unpaired t test. Comparisons between TTX + PE, PE and TTX are all significant (P < 0.001). P values for comparison between TTX + PE and PE are 0.7544 in somas and 0.7072 in processes, indicating no significant difference. (f) Schematic illustrating Ca2+ imaging of axon projections of GCaMP6f-expressing locus coeruleus (LC) neurons during running. Created with BioRender.com. (g, h) Fluorescence traces and the average amplitude of LC axonal Ca2+ activity in the motor cortex increased during running as compared to that during pre-run (n = 4 mice). Scale bar, 5 μm. (i, j) The average amplitude of running-induced dendritic Ca2+ changes with ACSF (i, n = 4 mice) or prazosin treatment (i, n = 5 mice); with saline (j, n = 5 mice) or DSP4 treatment (j, n = 4 mice). Two sided Mann-Whitney test. All P < 0.001. (k) Schematic of chemical depletion of norepinephrine with the DSP4 and astrocytic Ca2+ imaging during running. Created with BioRender.com. (l–o) Heatmaps (up) and average amplitude (below) of Ca2+ activity in somas (in orange) and processes (in blue) of astrocytes during running with saline (l&m, n = 6 mice) and DSP4 treatment (n&o, n = 6 mice). The color bar represents df/f0 within the ROI. The reason for the incomplete blockade of astrocytic Ca2+ activity after DSP4 could be attributed to the partial reduction of noradrenaline after DSP4 treatment61. (p–q) The average amplitude of astrocytic Ca2+ activity with saline and DSP4 treatment in the somas (p) and processes (q). Two sided Mann-Whitney test. P < 0.001, < 0.001. Boxes show the first quartile, median and third quartile of the stack data. Whiskers show minima and maxima without outliers. (r) Representative images (up) and amplitude (bottom) of PE-induced Ca2+ activity in both somas (WT: n = 143; IP3R2KO: n = 72) and processes (WT: n = 643; IP3R2KO: n = 780) of astrocytes during resting in WT and IP3R2KO mice. Scale bar, 20 μm. Two sided Mann-Whitney test. P < 0.001 for both somas and processes. Data are mean ± s.e.m. ns P > 0.05, ***P < 0.001.

Source data

Extended Data Fig. 3 Acute activation of astrocyte-specific Gq-DREADD induces transient Ca2+ fluctuations in astrocytes.

(a) Images representing 8 independent experiments of layer 1 astrocytes expressing human M3 muscarinic receptor Gq-DREADD (hM3Dq) and mcherry (asGq, in magenta) and/or cytosolic GCaMP6f Ca2+ indicators (in green) before and after CNO treatment. Co-expression of asGq and GCaMP6f are shown in white. Scale bar, 20μm. Following i.p. injection of CNO, transient Ca2+ fluctuations were visible in astrocyte network at 2 min, but were absent at 12 min under both resting and running conditions. (b–g) Heatmaps (up) and average amplitude (bottom) of somatic (in orange) and process (in blue) Ca2+ in astrocytes during resting 1-5 min post-CNO injection (b, c, rest_CNO 1-5 min, n = 5 mice), and during running before CNO injection (d, e, run_before CNO, n = 5 mice), 12 min post-CNO injection (f, g, run_CNO 12 min, n = 5 mice). The Color bar represents df/f0 within ROI. (h, i) The average amplitude of astrocytic Ca2+ transients increased post-CNO injection (CNO 1-5 min+rest) compared to control (before CNO+rest), and decreased 12 min post-CNO injection (CNO12min+run) compared to control (before CNO+run) in both somas (h, n = 264, 168; 148, 148) and processes (i, n = 1709, 2197; 1897, 1897) of astrocytes. Two sided Mann-Whitney test. All P < 0.001. (j) Average Ca2+ relative to 10% baseline_before CNO under quiet resting in both somas (in orange) and processes (in blue) of astrocytes before and after CNO activation of Gq-DREADD (somas: n = 318, 136, 549 respectively; processes: n = 2298, 1097, 3916 respectively). Data are mean ± s.e.m. One-way ANOVA. Soma: P = 0.9909, 0.3648, 0.6657 for Before CNO vs CNO 30 min, Before CNO vs CNO 1-4 h and CNO 30 min vs CNO1-4h respectively. Processes: P = 0.9579 for Before CNO vs CNO 30 min; P < 0.001 for both Before CNO vs CNO 1-4 h and CNO 30 min vs CNO1-4h. (k) Images representing 3 independent experiments of layer 1 astrocytic Ca2+ activity. (l, m) The average amplitude of astrocytic Ca2+ activity before and after CNO treatment in the somas (l, n = 106, 94 respectively) and processes (m, n = 754, 630 respectively) during motor running. Two sided paired t test. P = 0.9149 for somas; P = 0.9002 for processes. ns P > 0.05, ***P < 0.001.

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Extended Data Fig. 4 Sustained activation of both endogenous α1 and exogenous hM3Dq receptors results in the reduction of Ca2+ transients during motor running and the exhaustion of ER Ca2+ storage in astrocyte network.

(a) Images representing 4 independent experiments of running-induced astrocytic Ca2+ before and after 12 min of α1 agonist PE treatment. (b, c) The average amplitude of running-induced astrocytic Ca2+ activity before and after 12 min of PE treatment in the somas (b, n = 191, 210 respectively) and processes (c, n = 1190, 1232 respectively). Two sided Mann-Whitney test. All P < 0.001. Boxes show the first quartile, median and third quartile of the stack data. Whiskers show minima and maxima without outliers. (d) Schematic of the experimental design to sequentially activate endogenous (via PE) and exogenous Gq receptors (via CNO) in astrocytes expressing hM3Dq mcherry (asGq) and cytosolic GCaMP6f. (e) Images (up) and average amplitude (bottom) of astrocytic Ca2+ after CNO injection following 30 min post-PE treatment (1. CNO 1 min), and after PE application following 30 min post-CNO (2. PE 1 min) in both somas (left, n = 101, 110) and processes (right, n = 612, 1044) of astrocytes. Two sided Mann-Whitney test. P = 0.2903, 0.6547 for somas and processes respectively. (f) Images (up) of layer 1 astrocytes expressing hM3Dq (asGq-DREADD) and ER GCaMP sensors after saline injection, 30 min after PE bath application, and 1-5 min after CNO injection following 30 min of PE treatment. (bottom) The average amplitude of running-induced astrocytic ER Ca2+ changes after saline injection (control), 30 min after PE bath application, and 1-5 min after CNO injection following 30 min of PE treatment in the somas (n = 99, 101, 75 respectively) and processes (n = 2555, 3796, 2228 respectively). Two sided Mann-Whitney test. All P < 0.001. (g) Images (up) of astrocytes expressing hM3Dq (asGq-DREADD) and ER GCaMP sensors 10-15 min after saline injection, 30 min after CNO injection, and 1-5 min PE treatment following 30 min post-CNO injection. (bottom) The average amplitude of running-induced astrocytic ER Ca2+ changes after saline injection (control), 30 min after CNO injection, and 1–5 min of PE treatment following 30 min post-CNO injection in the somas (n = 330, 330, 179 respectively) and processes (n = 7631, 7631, 3785 respectively). These data indicate that both PE and asGq-DREADD activation eventually leads to the exhaustion of Ca2+ storage in the astrocyte ER, rendering astrocytes non-responsive to motor training. Two sided Mann-Whitney test. All P < 0.001. Data are mean ± s.e.m. ns P > 0.05, ***P < 0.001. Scale bar, 20 μm.

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Extended Data Fig. 5 The percentage of dendrites with the overall reduction of spine sizes after training is higher in mice with reduced astrocytic Ca2+.

(a–c) The average initial size in prazosin-treated (a, control: n = 5 mice, 92 spines, prazosin: n = 4 mice, 221 spines), IP3R2KO mice (b, control: n = 4 mice, 65 spines, IP3R2KO: n = 6 mice, 110 spines) and asGq-CNO mice (c, asGq-saline: n = 4 mice, 107 spines; asGq-CNO: n = 5 mice, 144 spines) and their respective control mice. Two sided Mann-Whitney test. P = 0.7628, 0.3187, 0.2887 for prazosin, IP3R2KO and asGq-CNO compared to their respective controls. Data are mean ± s.e.m. (d, e) The correlation between the initial size of spine and the subsequent spine size changes over 1.5 h of motor training in control (d, n = 264, P = 0.0207) and reduced astrocytic Ca2+ conditions (e, n = 475, P < 0.001). (f–h) The percentage of dendrites exhibiting spine size reduction over 1.5 h of motor training was higher in prazosin (f, control n = 5 mice, prazosin n = 4 mice), IP3R2KO (g, control n = 4 mice, IP3R2KO n = 6 mice) and asGq-CNO (h, control n = 4 mice, CNO n = 4 mice) mice as compared to their respective controls. Two sided Kolmogorove-Smirnov test. P = 0.3141, 0.0244, 0.0032 for prazosin treated, IP3R2KO and asGq-CNO mice compared to their respective controls. (i–k) Over 1.5 h of motor training, the reduction of astrocytic Ca2+ with either local application of prazosin (i, control n = 5 mice, prazosin n = 4 mice), IP3R2KO (j, control n = 4 mice, IP3R2KO n = 6 mice) or CNO activation of asGq (k, control n = 4 mice, CNO n = 4 mice) resulted in significantly larger percentage of tuft dendrites with more depotentiated spines. Two sided Kolmogorove-Smirnov test. P = 0.0086, 0.0302 and 0.0032 for prazosin treated, IP3R2KO and asGq-CNO mice compared to their respective controls. ns P > 0.05, *P < 0.05, **P < 0.01.

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Extended Data Fig. 6 Motor running-induced dendritic Ca2+ activity in control, prazosin-treated, IP3R2KO and asGq-DDREAD.

(a–c) Time intervals between two consecutive dendritic Ca2+ spikes over 2 min motor training were significantly shorter in prazosin (a, control 92 time intervals, 612 time intervals), IP3R2KO (b, control 62 time intervals, IP3R2KO 203 time intervals) and asGq-CNO (c, control 64 time intervals, asGq-CNO 160 time intervals) as compared to their respective controls. Two sided Kolmogorove-Smirnov test. P < 0.0001, = 0.0035, = 0.0076 for prazosin, IP3R2KO and asGq-CNO compared to their respective controls. (d–h) Dendrites with repetitive Ca2+ activity showed significantly higher integrated Ca2+ activity compared to dendrites with less repetitive Ca2+ activity in control (d, n = 4 mice, 12 dendrites), prazosin-treated (e, n = 4 mice, 37 dendrites), IP3R2KO (f, n = 6 mice, 25 dendrites), asGq-saline control (g, n = 3 mice, 19 dendrites) and asGq-CNO mice (h, n = 3 mice, 35 dendrites). Two sided Mann-Whitney test. P = 0.048, 0.0001, 0.0171, 0.0495, 0.0012 for control, prazosin, IP3R2KO, asGq-saline and asGq-CNO. Boxes show the first quartile, median and third quartile of the stack data. Whiskers show minima and maxima without outliers. (i–m) The number of Ca2+ spikes per individual dendrite over 2 min was significantly correlated with the accumulative dendritic Ca2+ activity in control (i, 12 dendrites), prazosin-treated (j, 37 dendrites), IP3R2KO (k, 25 dendrites), asGq-saline control (l, 19 dendrites) and asGq-CNO approach (m, 35 dendrites). P < 0.001, <0.001, =0.0029, =0.0011, <0.001 for control, prazosin, IP3R2KO asGq-saline, and asGq-CNO mice. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 7 Motor running-induced dendritic spine decreases in size with reduced astrocytic Ca2+.

(a, b) Distribution of spine size changes on less active dendrites (a, n = 67, 132) and more repetitive dendrites (b, n = 49, 135). Two sided Kolmogorove-Smirnov test. P < 0.001 for both conditions. (c) In control mice, the average of training-induced spine size increase was significantly higher on dendrites with less repetitive spikes compared to those with repetitive spikes (Less n = 67; repetitive: n = 49). Two sided Kolmogorove-Smirnov test. P = 0.0011. (d) Spines size changes on less active and repetitive dendrites under control (gray, n = 57, 26) and reduced astrocytic Ca2+ conditions (red, n = 67, 31). Two sided Mann-Whitney test. P = 0.071, P < 0.001. Data are mean ± s.e.m. ns P > 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 8 The latency from spine Ca2+ to dendritic Ca2+ activity predicts synaptic size changes.

(a) Schematic of latency from spine Ca2+ activity to dendritic Ca2+ activity. (b–f) Correlation between the latency from spine Ca2+ to dendritic Ca2+ activity and spine size changes in WT (b, n = 4mice, 31 spines), prazosin-treated (c, n = 3 mice, 36 spines), IP3R2KO (d, n = 6 mice, 43 spines), asGq-saline (e, n = 3 mice, 63 spines) and asGq-CNO mice (f, n = 3 mice, 58 spines). P = 0.002, < 0.001, = 0.0014, < 0.0001, = 0.0036 for control, prazosin, IP3R2KO asGq-saline, and asGq-CNO mice. (g) A larger fraction of spines exhibited their peak of spine Ca2+ activity prior to the peak of dendritic Ca2+ activity in prazosin-treated, IP3R2KO, and asGq-CNO mice as compared to the controls.

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Extended Data Fig. 9 Spine Ca2+ activity during the dendritic spiking window.

(a) Schematic of dendritic Ca2+ activity and co-active spine Ca2+ activity. (b–d) The percentage of spine active during dendritic activity window was higher in prazosin-treated (b, control 45 spines, prazosin 112 spines), IP3R2KO (c, control 54 spines, IP3R2KO 86 spines) and asGq-CNO mice (d, control 35 spines, asGq-CNO 80 spines). Two sided Mann-Whitney test. P = 0.053, 0.0012, 0.087 for prazosin, IP3R2KO and asGq-CNO compared to their controls. (e) Fluorescence traces of dendritic Ca2+ activity (black traces) and spine Ca2+ activity (grey traces) during motor training before (ACSF control) and after local application of CPA (adenosine A1 agonist) and DPCPX (adenosine A1 antagonist). (f, g) The percentage of spine active during dendritic activity window in CPA (f, control 69 spines, CPA 51 spines), DPCPX (g, control 99 spines, DPCPX 153 spine). Two sided Mann-Whitney test. P = 0.396, 0.0017 for CPA and DPCPX compared to their controls. All data are mean ± s.e.m. **P < 0.01.

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Extended Data Fig. 10 Treadmill running induces widespread elevation of astrocytic Ca2+ transients in different brain regions.

(a–d) Heatmaps of Ca2+ transients of astrocytes during the periods of pre-run and treadmill running in the motor cortex (n = 4 mice, 103 cells), prefrontal cortex (n = 3 mice, 113 cells), visual cortex (n = 4 mice, 101 cells) and hippocampus (n = 3 mice, 80 cells).

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Supplementary information

Reporting Summary

Supplementary Video 1

Motor training induces Ca2+ signaling in astrocytes. Ca2+ signals in astrocytes were readily detected using AAV-based GCaMP6 indicators. During resting period, local Ca2+ signals were observed. Upon motor training, Ca2+ transients emerged in the processes of some astrocytes and subsequently spread to their somas and adjacent astrocytes. Scale bar, 20 μm.

Supplementary Video 2

Ca2+ signaling among astrocyte network during motor training in transgenic mice expressing GCaMP5 and tdTomato. In transgenic mice with GLAST-CreER-dependent genetically encoded tdTomato and GCaMP5 indicators, nearly all GCaMP5-expressing astrocytes showed Ca2+ transients during motor training. Scale bar, 20 μm.

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Lai, B., Yuan, D., Xu, Z. et al. Astrocytic Ca2+ prevents synaptic depotentiation by limiting repetitive activity in dendrites during motor learning. Nat Neurosci (2025). https://doi.org/10.1038/s41593-025-02072-4

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