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Fear learning induces α7-nicotinic acetylcholine receptor-mediated astrocytic responsiveness that is required for memory persistence

Nature Neuroscience volume 24, pages 1686–1698 (2021)Cite this article

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Abstract

Memory persistence is a fundamental cognitive process for guiding behaviors and is considered to rely mostly on neuronal and synaptic plasticity. Whether and how astrocytes contribute to memory persistence is largely unknown. Here, by using two-photon Ca2+ imaging in head-fixed mice and fiber photometry in freely moving mice, we show that aversive sensory stimulation activates α7-nicotinic acetylcholine receptors (nAChRs) in a subpopulation of astrocytes in the auditory cortex. We demonstrate that fear learning causes the de novo induction of sound-evoked Ca2+ transients in these astrocytes. The astrocytic responsiveness persisted over days along with fear memory and disappeared in animals that underwent extinction of learned freezing behavior. Conditional genetic deletion of α7-nAChRs in astrocytes significantly impaired fear memory persistence. We conclude that learning-acquired, α7-nAChR-dependent astrocytic responsiveness is an integral part of the cellular substrate underlying memory persistence.

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Fig. 1: Sensory stimulation-evoked Ca2+ signaling in astrocytes of the mouse auditory cortex.
Fig. 2: nAchR dependence of footshock-evoked astrocytic responses.
Fig. 3: De novo induction of sound-evoked astrocytic responses by fear learning.
Fig. 4: Fear learning-dependent induction of astrocytic Ca2+ responsiveness in awake behaving mice.
Fig. 5: De novo induction of sound-evoked astrocytic Ca2+ responses by CS+ but not CS− in freely moving mice.
Fig. 6: Long-term persistence and extinction of astrocytic responsiveness.
Fig. 7: Impairment of fear memory persistence by tamoxifen-inducible deletion of the α7 subunit of nAChRs in astrocytes.
Fig. 8: Impairment of fear memory persistence by auditory cortex-specific deletion of astrocytic α7 subunit of nAChRs.

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Data availability

Any data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. No datasets that require mandatory deposition into a public database were generated during the current study. Source data underlying Figs. 1–8 and Extended Data Figs. 2–4 and 6–10 and Supplementary Figs. 5 and 6 are available as source data files. Source data are provided with this paper.

Code availability

No unique code was generated in this study. Source data are provided with this paper.

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Acknowledgements

We thank J. Lou for technical assistance. B.W. is a member of the Clinical Research Priority Program Molecular Imaging Network Zurich. This work was supported by the National Key R&D Program of China (no. 2018YFA0109600), the National Natural Science Foundation of China (no. 81771175) and the program of the China Scholarship Council (no. 201803170004) to K.Z., the National Natural Science Foundation of China (nos. 31925018, 31861143038, 31921003, 81671106) and Chongqing Basic Research grants (nos. cstc2019jcyjjqX0001 and cstc2019jcyj-cxttX0005) to X.C., the Deutsche Forschungsgemeinschaft (SFB870) and a European Research Council Advanced Grant to A.K. X.C. is a member of the CAS Center for Excellence in Brain Science and Intelligence Technology. A.K. is a Hertie Senior Professor of Neuroscience.

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Author notes

  1. These authors contributed equally: Kuan Zhang, Rita Förster.

Authors and Affiliations

  1. Brain Research Center and State Key Laboratory of Trauma, Burns, and Combined Injury, Third Military Medical University, Chongqing, China

    Kuan Zhang, Wenjing He, Jin Li, Chuanyan Yang, Meng Wang, Ran Ding, Ruijie Li, Tingliang Jian, Yanhong Wang, Jianxiong Zhang, Zhiqi Yang, Wenjun Jin & Xiaowei Chen

  2. Institute of Neuroscience, Technical University of Munich, Munich, Germany

    Rita Förster, Yonghai Zhang, Helmuth Adelsberger, Arthur Konnerth & Xiaowei Chen

  3. Munich Cluster for Systems Neurology (SyNergy) and Center for Integrated Protein Sciences, Munich, Germany

    Rita Förster, Yonghai Zhang, Helmuth Adelsberger & Arthur Konnerth

  4. Center for Neurointelligence, School of Medicine, Chongqing University, Chongqing, China

    Xiang Liao & Han Qin

  5. Advanced Institute for Brain and Intelligence, Guangxi University, Nanning, China

    Ruijie Li

  6. Department of Anatomy Histology and Embryology, Shanghai Medical College, Fudan University, Shanghai, China

    Song Qin

  7. Department of Anatomy Histology & Embryology, Air Force Medical University, Xi’an, China

    Yacheng Lu & Tao Chen

  8. Center for Neuron and Disease, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, China

    Tao Chen

  9. Institute of Pharmacology and Toxicology, University of Zurich and Neuroscience Center Zurich, Eidgenössische Technische Hochschule, Zurich, Switzerland

    Jillian Stobart & Bruno Weber

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Contributions

A.K. and X.C. designed the study. K.Z., R.F., W.H., J.L., C.Y., H.Q., M.W., R.D., R.L., T.J., Y.W., J.Z., Z.Y., Y.Z., J.S., B.W., H.A., A.K. and X.C. performed the main experiments. Y.L. and T.C. performed the immunoelectron microscopy experiments. S.Q. verified the astrocyte-specific Cre recombination of hGFAP-CreERT2:tdTomatoloxP/loxP mice. K.Z., R.F., J.L., C.Y., X.L., H.A. W.J. and X.C. performed the data analysis. A.K., X.C. and K.Z. wrote the manuscript with input from all coauthors.

Corresponding authors

Correspondence to Arthur Konnerth or Xiaowei Chen.

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Nature Neuroscience thanks Balazs Hangya and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sound-evoked Ca2+ signals in astrocytes of the mouse auditory cortex.

a, Two-photon fluorescence image of fluo-8AM-stained astrocytes in layer 2/3 of the mouse auditory cortex. The red arrowhead indicates an astrocyte that responded to sound (‘Sound responder’), while grey arrowheads indicate non-responding cells to sound (‘Sound non-responders’). b, Ca2+ traces (Δf/f) corresponding to the astrocytes indicated in panel a (sound stimuli indicated by grey bars; duration 9.9 s, pure tone of 8 kHz frequency, intensity 70 dB sound pressure level). Astrocytes were defined as ‘responders’ (Fig. 1 and Extended Data Fig. 1) when the maxima of averaged Ca2+ signals from 3 consecutive trials 0-9 s after stimuli were above 3 × s.d. of baseline. Therefore, the ‘non-responders’ could include occasional weak responses that were smaller than 3 × s.d of baseline traces.

Extended Data Fig. 2 Footshock-induced astrocytic Ca2+ transients involve Ca2+ release from internal stores and are blocked by TTX, but not require gap junctions, mAChRs, mGluRs or iGluRs in the adult mouse cortex.

a, b, Representative cells showing footshock-induced astrocytic Ca2+ signals before and after application of thapsigargin (a) or ryanodine (b). c, Footshock-induced Ca2+ transients in astrocytes of auditory cortex without or with a gap junction blocker carbenoxolone. Left, two-photon image of fluo-8AM labelled astrocytes. Right, representative footshock-evoked Ca2+ transients in astrocytes indicated in the left panel. d, Summary results of the carbenoxolone experiments. Note a minor but no significant reduction in the amplitude of footshock-evoked Ca2+ transients in the presence of carbenoxolone (control versus carbenoxolone, Z = 1.6499, P = 0.0990; n = 25 cells from 4 mice, two-sided Wilcoxon signed-rank test). e, Two-photon image of fluo-8AM-stained astrocytes in layer 2/3 of the mouse auditory cortex. f, Footshock-induced Ca2+ transients in astrocytes, outlined in panel a, before and after local application of TTX (0.5 µM). g, Summary of the TTX experiments (control versus TTX, Z = 6.4515, P = 1.1076 E-10; ***P < 0.001, two-sided Wilcoxon signed-rank test, n = 55 cells from 5 mice). h, Representative cells showing footshock-induced astrocytic Ca2+ transients without or with mAChR antagonist VU0255035. i, Representative cells showing footshock-induced astrocytic Ca2+ transients without or with the combination of group I mGluR antagonists MPEP and LY367385. j, Representative cells showing footshock-induced astrocytic Ca2+ transients without or with the combination of iGluR (ionotropic glutamate receptor) antagonists APV and CNQX. All data in the figure are shown as mean ± s.e.m.

Source data

Extended Data Fig. 3 Footshock stimulation-evoked astrocytic Ca2+ signals in the auditory cortex of anaesthetized and awake mice.

a, Two-photon image of labeled astrocytes in layer 2/3 of the mouse auditory cortex. b, Footshock-evoked Ca2+ transients (Δf/f) from the astrocytes indicated in panel a in anaesthetized (left) and awake (right) states from the same imaging plane in the same mouse (footshock stimuli indicated by red dashed boxes). Awake recordings were achieved at least 1 hour after stopping isoflurane application. c, Bar graphs summarizing the onset latencies of astrocytic Ca2+ transients during anaesthetized and awake states. n = 101 cells from 5 mice in the anaesthetized group; n = 51 cells from 4 mice in the awake group (anaesthetized versus awake, Z = 1.2097, P = 0.2264, two-sided Wilcoxon rank-sum test). All data in the figure are shown as mean ± s.e.m.

Source data

Extended Data Fig. 4 Neither α-adrenergic receptors nor α1-adrenergic receptors are required for footshock-induced astrocytic Ca2+ transients.

a, Representative cells showing footshock-induced astrocytic Ca2+ transients without or with the α-adrenergic receptor antagonist phentolamine. b, Summary of the amplitudes of footshock-evoked astrocytic responses and the fractions of responding cells in the presence of phentolamine. No significant effect was observed (left panel: the number of cells tested is indicated on the top of each bar. Data are from 3 mice; control versus phentolamine, Z = -1.4206, P = 0.1554; right panel: n = 9 fields of view, 41 cells from 3 mice, n = 9 fields of view, 22 cells from 3 mice; control versus Phentolamine, Z = -0.1349, P = 0.8927; two-sided Wilcoxon rank-sum test). All recordings in this figure were performed in the auditory cortex of head-fixed, awake mice. c, Footshock-induced astrocytic Ca2+ transients without or with the α-1 adrenergic receptor antagonist prazosin in 5 representative cells. d, Summary of the amplitudes of footshock-evoked astrocytic responses and the fractions of responding cells in the presence of prazosin. No significant effect was observed (left panel: the number of cells is indicated in each bar graph. Data are from 4 mice; control versus prazosin, Z = 0.7183, P = 0.4725; right panel: n = 6 fields of view, 64 cells from 4 mice in control group; n = 6 fields of view, 62 cells from 4 mice in prazosin group; control versus prazosin, Z = 0.0000, P = 1.0000; two-sided Wilcoxon rank-sum test). All recordings in this figure were performed in the auditory cortex. All data in the figure are shown as mean ± s.e.m.

Source data

Extended Data Fig. 5 Specific expression α7-nicotinic receptors in those astrocytes that respond to nicotine application.

a, In vivo two-photon imaging of astrocyte response to local nicotine application. Left, an imaging plane showing 3 astrocytes labelled with rhod-2AM. The dashed lines indicate the position of an electrode for nicotine application. Right, nicotine-evoked astrocytic Ca2+ transient in cell1 but not in cell2 and cell3. b, Electroporation of two nearby neurons (N1 and N2) in the same imaging plane with OGB-1 (green) as markers. Left, green channel showing OGB-1-labeled neurons. Right, red and green channels showing both rhod-2 AM-labeled astrocytes and OGB-1-labeled neurons. c, Immunostaining of α7 receptors. Post-hoc histology was performed after in vivo two-photon imaging. Note the expression of α7 receptors in cell1 but not in cell2 and cell3, consistent with the in vivo two-photon imaging result. d, Summary of all astrocytes tested, showing that 5 astrocytes responding to nicotine (responder) also express α7 receptors, while 7 non-responders express no α7 receptors. These experiments were repeated in 3 mice.

Extended Data Fig. 6 Absence of sound-evoked astrocytic Ca2+ signals following trace fear conditioning.

a, Summary of the freezing levels after trace fear conditioning (n = 13 mice; baseline versus CS+, Z = −3.1798, P = 0.0015, two-sided Wilcoxon signed rank test, **P < 0.01.). b, Ca2+ traces from astrocytes that were not responsive to sound stimulation before (left) and after (right) trace fear conditioning (black traces: two consecutive sound stimuli). c, Bar graph summarizing of the amplitude of astrocytic Ca2+ transients in response to sound stimulation before and after trace fear conditioning. Data of ‘before’ group is the same as the ‘control’ group in Fig. 6i. The numbers of cells (and mice) are indicated in each bar graph (before versus after, Z = 0.2550, P = 0.7987, Two-sided Wilcoxon rank-sum test). d, Bar graph summarizing of the fraction of astrocytes responding to sound stimulation before and after trace fear conditioning (before: 1.42 ± 0.78 %, after: 1.67 ± 1.14%; before versus after, Z = 0.058, P = 0.9538, Two-sided Wilcoxon rank-sum test). Data of ‘before’ group is the sum of the ‘control’ group in Fig. 6i. The numbers of cells (and mice) are indicated in each bar graph. For this trace fear conditioning experiment, the trace interval separating CS+ and US was 20 s. Five pairings were delivered for conditioning and the interval between two pairings was 3 min. All data in the figure are shown as mean ± s.e.m.

Source data

Extended Data Fig. 7 De novo induction of sound-evoked astrocytic Ca2+ responses by fear conditioning in the hippocampus (CA1 and dorsal hippocampus, dHP).

a, Confocal images of astrocytes labeled by AAV5-GfaABC1D-cytoGCaMP6f-SV40 (green) in the CA1 region. b, Astrocytic Ca2+ transients in response to footshock or sound before, during and after pairing in the CA1 region. c, Bar graphs summarizing the amplitudes of astrocytic Ca2+ transients in response to footshock (left) or sound (right) before and after conditioning in the CA1 region. n = 6 mice (left, footshock: before versus after, Z = 0.1348, P = 0.8927; right, sound: before versus after, Z = −2.2014, P = 0.0277; *P < 0.05, two-sided Wilcoxon signed rank test). d, Summary of the freezing levels before and after fear conditioning. n = 6 mice (baseline: before versus after, Z = −0.9439, P = 0.3452; CS: before versus after, Z = −2.2014, P = 0.0277; *P < 0.05, two-sided Wilcoxon signed rank test). e, Confocal images of astrocytes labeled by AAV5-GfaABC1D-cytoGCaMP6f-SV40 (green) in the dHP. f, Astrocytic Ca2+ transients in response to footshock or sound before, during and after pairing in the dHP. g, Bar graphs summarizing the amplitudes of astrocytic Ca2+ transients in response to footshock (left) or sound (right) before and after conditioning in the dHP. n = 6 mice (left, footshock: before versus after, Z = 1.1531, P = 0.2489; right, sound: before versus after, Z = −2.2014, P = 0.0277; *P < 0.05, two-sided Wilcoxon signed rank test). h, Summary of the freezing levels before and after fear conditioning. n = 7 mice (baseline: before versus after, Z = −0.8452, P = 0.3980; CS: before versus after, Z = −2.3664, P = 0.0180; *P < 0.05, two-sided Wilcoxon signed rank test). All data in the figure are shown as mean ± s.e.m.

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Extended Data Fig. 8 De novo induction of sound-evoked Ca2+ astrocytic responses by fear conditioning in the basolateral amygdala (BLA), but not in the somatosensory cortex.

a, Confocal images of astrocytes labeled by AAV5-GfaABC1D-cytoGCaMP6f-SV40 (green) in the BLA. b, Astrocytic Ca2+ transients in response to footshock or sound before, during and after pairing in the BLA of amygdala. c, Bar graphs summarizing the amplitudes of astrocytic Ca2+ transients in response to footshock (left) or sound (right) before and after conditioning in the BLA. n = 5 mice (left, footshock: before versus after, Z = 1.4832, P = 0.1380; right, sound: before versus after, Z = −2.0226, P = 0.0431; *P < 0.05, two-sided Wilcoxon signed rank test). d, Summary of the freezing levels before and after fear conditioning. n = 7 mice (baseline: before versus after, Z = −1.5724, P = 0.1159; CS: before versus after, Z = −2.3664, P = 0.0180; *P < 0.05, two-sided Wilcoxon signed rank test). e, Confocal imaging of astrocytes labeled by AAV5-GfaABC1D-cytoGCaMP6f-SV40 (green) and S100β (red) in the somatosensory cortex. f, Astrocytic Ca2+ transients in response to footshock or sound before, during and after pairing in the somatosensory cortex. g, Bar graphs summarizing the amplitudes of astrocytic Ca2+ transients in response to footshock (left) or sound (right) before and after conditioning in the somatosensory cortex. n = 7 mice (left, footshock: before versus after, Z = 0.1690, P = 0.8658; right, sound: before versus after, Z = −1.0142, P = 0.3105; two-sided Wilcoxon signed rank test). h, Summary of the freezing levels before and after fear conditioning. n = 6 mice (baseline: before versus after, Z = −1.5724, P = 0.1159; CS: before versus after, Z = −2.2014, P = 0.0277; *P < 0.05, two-sided Wilcoxon signed rank test). All data in the figure are shown as mean ± s.e.m.

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Extended Data Fig. 9 Characterization of tamoxifen-inducible astrocytic α7-nAchR conditional knockout mice (hGFAP-CreERT2/Chrna7loxp/loxp, cKO).

a, PCR analysis of hGFAP-CreERT2:Chrna7WT/WT (cWT) and hGFAP-CreERT2:Chrna7loxp/loxp mice (α7-cKO mice). The experiment was repeated before immunostaining and two-photon imaging experiment. b, c, α7-nAchR immunostaining in the auditory cortex of a cWT (b) or a cKO (c) mouse indicates a pronounced loss of α7-nAchR-positive astrocytes in cKO. Left, neuronal staining (neuronal marker: NeuN) in L1 and L2/3 in the auditory cortex. Right, astrocyte staining (astrocyte marker: S100β) in the auditory cortex. One astrocyte from panel b was shown in Fig. 7b in an expanded scale. d, Bar graphs summarizing the α7-nAchR expression in neurons in the auditory cortex of cWT and cKO mice. n = 12 sections from 4 mice in each group (cWT versus cKO, Z = −0.6067, P = 0.5440, two-sided Wilcoxon rank-sum test). e, Bar graphs summarizing the α7-nAchR expression in astrocytes in the auditory cortex of cWT and cKO mice. n = 16 sections from 5 mice in cWT group; n = 11 sections from 4 mice in cKO group. (cWT versus cKO, Z = 4.3257, P = 1.52 E-5, ***P < 0.001, two-sided Wilcoxon rank-sum test). f, In vivo two-photon Ca2+ imaging of L1 interneurons in the auditory cortex of a cKO mouse, with dashed lines indicating neurons of interest. g, Single trials and their average of Ca2+ responses to footshock from these neurons as indicated in panel f. h, Bar graph summarizing the fraction of L1 neurons that responded to footshock in cWT and cKO mice. n = 9 fields of view, 42 cells from 3 mice in cWT group; n = 15 fields of view, 127 cells from 6 mice in cKO group (cWT versus cKO, Z = 0.2684, P = 0.7884; two-sided Wilcoxon rank-sum test). All data in the figure are shown as mean ± s.e.m.

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Extended Data Fig. 10 Characterization of virus-induced region-specific astrocytic α7-nAchR conditional knockout mice (AAV + Chrna7loxp/loxp, cKO).

a, Post-hoc image showing the position of AAV-transfected region in the extended auditory cortex. b, Neuron (NeuN) and α7-nAchR staining in L1 and L2/3 in the auditory cortex of a cWT (upper; AAV5-GfaABC1D-Cre was injected into the auditory cortex of Chrna7WT/WT mice) or a cKO (lower; AAV5-GfaABC1D-Cre was injected into the auditory cortex of Chrna7loxP/loxP mice) mouse. c, α7-nAchR and S100β immunostaining in the auditory cortex of a cWT (upper) or a cKO (lower) mouse, indicating a pronounced loss of α7-nAchR-positive astrocytes in cKO. d, High-magnification images showing immunostaining of α7-nAChRs and S100β in cWT (left) and cKO (right) as indicated by the white boxes in panel c. e, Bar graphs summarizing the α7-nAchR expression in neurons in the auditory cortex of cWT and cKO mice. n = 8 sections from 4 mice in each group (cWT versus cKO, Z = 0.3153, P = 0.7525, two-sided Wilcoxon rank-sum test). f, Bar graphs summarizing the α7-nAchR expression in astrocytes in the auditory cortex of cWT and cKO mice. n = 10 sections from 4 mice in each group. (cWT versus cKO, Z = 3.8675, P = 1.10 E-4, ***P < 0.001, two-sided Wilcoxon rank-sum test). g, In vivo two-photon Ca2+ imaging of L1 interneurons in the auditory cortex of an AAV-induced cKO mouse, with dashed lines indicating neurons of interest. h, Single trials and their average of Ca2+ responses to footshock from these neurons as indicated in panel g. i, Bar graph summarizing the fraction of L1 neurons that responded to footshock in cWT and cKO mice. n = 9 fields of view, 45 cells from 3 mice in cWT group; n = 9 fields of view, 52 cells from 3 mice in cKO group (cWT versus cKO, Z = 0.1766, P = 0.8598, two-sided Wilcoxon rank-sum test). All data in the figure are shown as mean ± s.e.m.

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Zhang, K., Förster, R., He, W. et al. Fear learning induces α7-nicotinic acetylcholine receptor-mediated astrocytic responsiveness that is required for memory persistence. Nat Neurosci 24, 1686–1698 (2021). https://doi.org/10.1038/s41593-021-00949-8

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