Abstract
The physical manifestations of memory formation and recall are fundamental questions that remain unresolved1. At the cellular level, ensembles of neurons called engrams are activated by learning events and control memory recall1,2,3,4,5. Astrocytes are found in close proximity to neurons and engage in a range of activities that support neurotransmission and circuit plasticity6,7,8,9,10. Moreover, astrocytes exhibit experience-dependent plasticity11,12,13, although whether specific ensembles of astrocytes participate in memory recall remains obscure. Here we show that learning events induce c-Fos expression in a subset of hippocampal astrocytes, and that this subsequently regulates the function of the hippocampal circuit in mice. Intersectional labelling of astrocyte ensembles with c-Fos after learning events shows that they are closely affiliated with engram neurons, and reactivation of these astrocyte ensembles stimulates memory recall. At the molecular level, learning-associated astrocyte (LAA) ensembles exhibit elevated expression of nuclear factor I-A, and its selective deletion from this population suppresses memory recall. Taken together, our data identify LAA ensembles as a form of plasticity that is sufficient to provoke memory recall and indicate that astrocytes are an active component of the engram.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
£17.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
£199.00 per year
only £3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
RNA-sequencing data have been deposited at the NCBI GEO under accession number GSE254016. All other data are available from the corresponding author on reasonable request. Source data are provided with this paper.
References
Josselyn, S. A. & Tonegawa, S. Memory engrams: recalling the past and imagining the future. Science 367, eaaw4325 (2020).
Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).
Ramirez, S. et al. Creating a false memory in the hippocampus. Science 341, 387–391 (2013).
Roy, D. S. et al. Brain-wide mapping reveals that engrams for a single memory are distributed across multiple brain regions. Nat. Commun. 13, 1799 (2022).
Han, J.-H. et al. Selective erasure of a fear memory. Science 323, 1492–1496 (2009).
Dallérac, G., Zapata, J. & Rouach, N. Versatile control of synaptic circuits by astrocytes: where, when and how? Nat. Rev. Neurosci. 19, 729–743 (2018).
Kofuji, P. & Araque, A. Astrocytes and behavior. Annu. Rev. Neurosci. 44, 49–67 (2021).
Allen, N. J. & Eroglu, C. Cell biology of astrocyte–synapse interactions. Neuron 96, 697–708 (2017).
Nagai, J. et al. Behaviorally consequential astrocytic regulation of neural circuits. Neuron 109, 576–596 (2021).
Soto, J. S. et al. Astrocyte–neuron subproteomes and obsessive–compulsive disorder mechanisms. Nature 616, 764–773 (2023).
Sardar, D. et al. Induction of astrocytic Slc22a3 regulates sensory processing through histone serotonylation. Science 380, eade0027 (2023).
Cheng, Y.-T. et al. Social deprivation induces astrocytic TRPA1–GABA suppression of hippocampal circuits. Neuron 111, 1301–1315 (2023).
Lawal, O., Ulloa Severino, F. P. & Eroglu, C. The role of astrocyte structural plasticity in regulating neural circuit function and behavior. Glia 70, 1467–1483 (2022).
Fleischmann, A. et al. Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS. J. Neurosci. 23, 9116–9122 (2003).
Katche, C. et al. Delayed wave of c-Fos expression in the dorsal hippocampus involved specifically in persistence of long-term memory storage. Proc. Natl Acad. Sci. USA 107, 349–354 (2010).
Lacagnina, A. F. et al. Distinct hippocampal engrams control extinction and relapse of fear memory. Nat. Neurosci. 22, 753–761 (2019).
Khakh, B. S. & Sofroniew, M. V. Diversity of astrocyte functions and phenotypes in neural circuits. Nat. Neurosci. 18, 942–952 (2015).
Papouin, T., Dunphy, J., Tolman, M., Foley, J. C. & Haydon, P. G. Astrocytic control of synaptic function. Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20160154 (2017).
Endo, F. et al. Molecular basis of astrocyte diversity and morphology across the CNS in health and disease. Science 378, eadc9020 (2022).
Kol, A. et al. Astrocytes contribute to remote memory formation by modulating hippocampal–cortical communication during learning. Nat. Neurosci. 23, 1229–1239 (2020).
Cheng, Y.-T. et al. Inhibitory input directs astrocyte morphogenesis through glial GABABR. Nature 617, 369–376 (2023).
Henneberger, C., Papouin, T., Oliet, S. H. R. & Rusakov, D. A. Long-term potentiation depends on release of d-serine from astrocytes. Nature 463, 232–236 (2010).
Huang, A. Y.-S. et al. Region-specific transcriptional control of astrocyte function oversees local circuit activities. Neuron 106, 992–1008 (2020).
Adamsky, A. et al. Astrocytic activation generates de novo neuronal potentiation and memory enhancement. Cell 174, 59–71 (2018).
Suthard, R. L. et al. Basolateral amygdala astrocytes are engaged by the acquisition and expression of a contextual fear memory. J. Neurosci. https://doi.org/10.1523/JNEUROSCI.1775-22.2023 (2023).
Yap, E.-L. & Greenberg, M. E. Activity-regulated transcription: bridging the gap between neural activity and behavior. Neuron 100, 330–348 (2018).
Gleichman, A. J., Kawaguchi, R., Sofroniew, M. V. & Carmichael, S. T. A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences. Nat. Commun. 14, 7426 (2023).
Sun, W. et al. Spatial transcriptomics reveal neuron–astrocyte synergy in long-term memory. Nature https://doi.org/10.1038/s41586-023-07011-6 (2024).
Chen, M. B., Jiang, X., Quake, S. R. & Südhof, T. C. Persistent transcriptional programmes are associated with remote memory. Nature 587, 437–442 (2020).
Yu, X. et al. Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior. Neuron 99, 1170–1187 (2018).
Ung, K., Tepe, B., Pekarek, B., Arenkiel, B. R. & Deneen, B. Parallel astrocyte calcium signaling modulates olfactory bulb responses. J. Neurosci. Res. 98, 1605–1618 (2020).
Choi, J.-H. et al. Interregional synaptic maps among engram cells underlie memory formation. Science 360, 430–435 (2018).
Park, H. et al. Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors. Mol. Brain 8, 7 (2015).
Yu, X. et al. Context-specific striatal astrocyte molecular responses are phenotypically exploitable. Neuron https://doi.org/10.1016/j.neuron.2020.09.021 (2020).
Lin, C.-C. J. et al. Identification of diverse astrocyte populations and their malignant analogs. Nat. Neurosci. 20, 396–405 (2017).
Morel, L. et al. Molecular and functional properties of regional astrocytes in the adult brain. J. Neurosci. 37, 8706–8717 (2017).
de Ceglia, R. et al. Specialized astrocytes mediate glutamatergic gliotransmission in the CNS. Nature https://doi.org/10.1038/s41586-023-06502-w (2023).
Ghandour, K. et al. Orchestrated ensemble activities constitute a hippocampal memory engram. Nat. Commun. 10, 2637 (2019).
Ung, K. et al. Olfactory bulb astrocytes mediate sensory circuit processing through Sox9 in the mouse brain. Nat. Commun. 12, 5230 (2021).
Rao-Ruiz, P. et al. Engram-specific transcriptome profiling of contextual memory consolidation. Nat. Commun. 10, 2232 (2019).
Lozzi, B., Huang, T.-W., Sardar, D., Huang, A. Y.-S. & Deneen, B. Regionally distinct astrocytes display unique transcription factor profiles in the adult brain. Front. Neurosci. https://doi.org/10.3389/fnins.2020.00061 (2020).
Cheng, Y.-T., Woo, J. & Deneen, B. Sculpting astrocyte diversity through circuits and transcription. Neuroscientist https://doi.org/10.1177/10738584221082620 (2022).
Scavuzzo, M. A. et al. Pancreatic cell fate determination relies on Notch ligand trafficking by NFIA. Cell Rep. 25, 3811–3827 (2018).
Srinivasan, R. et al. Ca2+ signaling in astrocytes from Ip3r2−/− mice in brain slices and during startle responses in vivo. Nat. Neurosci. 18, 708–717 (2015).
Ewels, P., Magnusson, M., Lundin, S. & Käller, M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047–3048 (2016).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Acknowledgements
This work was supported by US National Institutes of Health grants R35-NS132230, R21-MH134002 and R01-AG071687 to B.D. M.R.W. is supported by American Heart Association grant AHA-23POST1019413. W.K. is supported by the National Research Foundation of Korea (RS-2024-00405396). We thank the David and Eula Wintermann Foundation for support. We thank the Optogenetics and Viral Vectors/Neuroconnectivity Core (supported by US National Institutes of Health grant U54-HD08309) at the Jan and Dan Duncan Neurological Research Institute for virus production. This research was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under award P50HD103555 for use of the Microscopy Core facilities and the Animal Phenotyping & Preclinical Endpoints Core facilities. The schematics in the figures were created using Biorender.com.
Author information
Authors and Affiliations
Contributions
W.K., M.R.W. and B.D. conceived the project and designed the experiments. W.K. and M.R.W. generated the AAV viral constructs and performed the brain injections, behavioural studies, analysis of brain tissue and RNA-seq. J.W. did the electrophysiological recordings. Y.K., K.Y., S.M. and D.S. assisted with the RNA-seq studies and bioinformatics analysis. K.Y., E.M. and Y.K. assisted with analysis and imaging of brain tissue. M.R.W., W.K. and B.D. wrote the manuscript. For CV purposes, M.R.W. and W.K. can be interchanged in the author list because they contributed equally, with the order decided by tossing a coin.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks C. Justin Lee, Gertrudis Perea and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Chemogenetic activation of hippocampal neurons induces c-Fos expression in a subset of astrocytes.
a. Genetic system and timeline for chemogenetic activation of dentate gyrus neurons. b. Representative image of hM3D-mCherry labeling in the hippocampus (representative of 3 mice). c. Representative immunostaining of c-Fos expression in hippocampal astrocytes. d. Quantification of c-Fos+ Sox9+ astrocytes following saline or CNO injection (n = 4 mice per group, upper panel: two-tailed t test, ****P < 0.0001, lower panel: two-sided Welch’s corrected t test, **P = 0.003). e. Genetic system and timeline for chemogenetic activation of excitatory dentate gyrus neurons. Retrograde AAV-hSyn-hM4D-mCherry was used to inhibit inputs. f. Representative immunostaining of c-Fos expression in hippocampal astrocytes. g. Quantification of c-Fos+ Sox9+ astrocytes following CNO injection (n = 5 mice per group, upper panel: one-way ANOVA (P < 0.0001) and Tukey tests, ****P < 0.0001, lower panel: one-way ANOVA (P < 0.0001) and Tukey tests, ***P = 0.0002, ****P < 0.0001). Data are mean ± SEM.
Extended Data Fig. 2 Specific targeting of astrocytes with AAV-GFAP-Cre-4x6T.
a. Schematic of genetic system for testing specificity of AAV-GFAP-Cre-4x6T in tdTomato Cre reporter mice. b. Representative immunostaining for Sox9+ astrocytes and NeuN+ neurons. c. Quantification of overlap between tdTomato and Sox9 or NeuN (n = 8 mice). Data are mean ± SEM. Panel a was created using Biorender.com.
Extended Data Fig. 3 Additional characterization of c-Fos expression and Fos cKO.
a. Representative images of c-Fos expression in hippocampal neurons from mice left in homecage or 90 min after fear conditioning. b. Quantification of neuronal c-Fos expression (n = 4 mice per group (HC = homecage, FC = fear conditioning), two-tailed t tests, **P = 0.002 (upper), 0.008 (lower). c. Representative images encompassing portions of stratum radiatum, stratum lacunosum, and stratum moleculare of Sox9 and c-Fos. Arrows indicate c-Fos+ Sox9+ cells. Representative of 4 mice/group. d. Representative images showing astrocytic expression of Cre following injection of AAV-GFAP-Cre-4x6T. Images were processed with a dehaze filter. Representative of 4 mice. e. Representative images of astrocytic c-Fos expression from GFAP-mCherry and GFAP-Cre-4x6T injected mice. Arrows indicate c-Fos+ Sox9+ cells. Representative of 8 mice/group. Data are mean ± SEM.
Extended Data Fig. 4 Calcium imaging and electrophysiology in Fos cKO mice.
a. Calcium signal traces from control and Fos cKO astrocytes. Each row in the heatmaps represents a single astrocyte (n = 20 control, 18 Fos cKO astrocytes, n = 3 mice per group). b-c. Quantification of calcium signaling parameters in soma/major banches (b) and microdomains (c) (n = 20 control, 18 Fos cKO astrocytes, n = 3 mice per group for some/main branches; n = 16 astrocytes, n = 3 mice per group for microdomains). Two-tailed t test, *P = 0.038. d. Representative traces and quantification of sEPSCs in control (n = 8) and Fos cKO (n = 10) mice. Two-tailed t test, *P = 0.026. e. Representative traces and quantification of sIPSCs in control (n = 8) and Fos cKO (n = 9) mice. Data are mean ± SEM.
Extended Data Fig. 5 Additional characterization of learning-associated astrocyte distribution and microdomain calcium activity.
a. Quantification of learning-associated astrocytes across hippocampal layers (see Fig. 2). Comparisons on graph are P values from Dunnett’s tests. N = 6 homecage, N = 5 context only, N = 5 0.75 mA shock, N = 8 1.5 mA shock mice. b. Representative images of tdTomato+ learning-associated astrocytes in the hippocampus and quantification of co-labeling with GFAP. Str. oriens = stratum oriens, str. pyr. = stratum pyramidale, str. rad. = stratum radiatum, str. l-m. = stratum lacunosum-moleculare, str. mol. = stratum moleculare of the dentate gyrus, str. gr. = stratum granulosum. Representative of 5 mice each. c. Quantification of microdomain calcium activity parameters related to Fig. 2d,e. Middle panel: two-tailed Mann Whitney test, *P = 0.030. Right panel: two-tailed t test, *P = 0.036. n = 16 homecage and 17 fear conditioning cells from 3 mice per group. Data are mean ± SEM.
Extended Data Fig. 6 Tamoxifen does not affect astrocytic c-Fos expression.
a. Timeline for examining astrocytic c-Fos expression after injection of tamoxifen, 4-hydroxytamoxifen, or vehicle. b. Representative images of Sox9 and c-Fos immunostaining in CA1 and DG. c. Quantification of c-Fos expression in Sox9+ cells. One-way ANOVA, P ≥ 0.268. n = 4 mice per group. Data are mean ± SEM.
Extended Data Fig. 7 Tagging learning-associated astrocyte ensembles with Fos-FlpER.
a. Schematic of genetic system and timeline for evaluating activity-dependent labeling of astrocytes with Fos-FlpER. b. Representative images of hippocampal astrocytes labeled in home cage and fear conditioned mice. c. Quantification of tdTomato+ cells (n = 4 mice per group, two-tailed t test, **P = 0.001). d. Representative immunostaining showing colocalization of tdTomato with GFAP. Representative of 4 mice/group. Data are mean ± SEM.
Extended Data Fig. 8 Learning-associated astrocytes re-express c-Fos after memory recall.
a. Schematic of genetic system for labeling learning-associated astrocytes with tdTomato and experimental design for labeling active astrocytes during fear conditioning or recall. b. Representative images of tdTomato+ learning associated astrocytes. c. Quantification of density of tdTomato+ astrocytes tagged during fear conditioning (FC) or recall (N = 4 mice per group). Two-tailed t test, P = 0.818. d. Schematic of genetic system for labeling learning-associated astrocytes with tdTomato during fear conditioning and experimental design for examining c-Fos expression after memory recall. e. Representative image of c-Fos expression in a learning-associated astrocyte after memory recall. Arrow indicates a c-Fos+ tdTomato+ astrocyte. f. Quantification of c-Fos expression after recall in Sox9+ non-learning associated astrocytes (tdTomato-) and learning-associated astrocytes following recall (tdTomato+). tdTomato+ astrocytes were ~2.4% of all Sox9+. N = 4 mice, two-tailed Welch’s t test, **P = 0.002. Data are mean ± SEM.
Extended Data Fig. 9 Learning-associated astrocytes facilitate long-term potentiation.
a. Schematic of genetic system for expressing hM3D-mCherry in learning-associated astrocytes. b. Experimental timeline and schematic of LTP recordings. c. fEPSP traces from slices treated with CNO or saline followed by subthreshold stimulation (n = 6 fear conditioned hM3D + saline, n = 9 homecage hM3d + CNO, n = 7 fear conditioned hM3D + CNO, n = 7 pan-astrocyte hM3D + CNO mice). d. Summary of fEPSP slope from the last 5 min of recordings in panel c (n = 6 fear conditioned hM3D + saline, n = 9 homecage hM3d + CNO, n = 7 fear conditioned hM3D + CNO, n = 7 pan-astrocyte hM3D + CNO mice). One way ANOVA, P = 0.004; Dunnett’s tests, *P = 0.047, **P = 0.007. e. fEPSP traces from slices treated with CNO but without electrical stimulation. Two-tailed t tests comparing 5-minute time bins, *P ≤ 0.049. n = 9 LAA hM3D and 8 mCherry mice. Data are mean ± SEM. Panel a, b were created using Biorender.com.
Extended Data Fig. 10 Additional characterization of learning-associated astrocyte-engram neuron interactions and viral labeling.
a. Schematic of AAVs and experimental timeline related to Fig. 3k–m. b. Images showing AAV-mediated labeling and immunostaining for c-Fos in dentate gyrus. Arrow indicates an EYFP+ c-Fos positive engram neurons. Arrowhead indicates an hM3D-mCherry+ astrocyte located within the dendritic arbor of the neuron. The magenta channel fluorescence was subtracted from the red channel in order to offset spectral bleed-through. Representative of 4 mice. c. Re-activation of engram neurons in dentate gyrus was increased by learning-associated astrocyte activation (two-tailed t test, **P = 0.007). n = 10 saline, 7 CNO mice. d. Low magnification images showing AAV-mediated labeling of engram neurons and immunostaining for c-Fos. e. Images of mCherry and EYFP labeled cells in CA1 and DG. Representative of 4 mice. f. Quantification of the ratio of engram neurons (EYFP+) to learning-associated astrocytes (mCherry+) in CA1 and DG. N = 3 mice per region. g. Low magnification image showing viral targeting of the hippocampus with AAV-GFAP-mCherry. Representative of 8 mice. h. Higher magnification image of viral labeling representative of 6 mice. i. Quantification of viral labeling of astrocytes. n = 6 mice per region. Data are mean ± SEM.
Extended Data Fig. 11 Remote reactivation of learning-associated astrocyte ensembles elicits recall.
a. Timeline. b. Schematic of genetic system for evaluating recall after remote reactivation of the fear-tagged astrocyte ensemble. c. Quantification of freezing behavior prior to foot shocks (n = 8 saline, n = 7 CNO mice, two-tailed t test, P = 0.368). d. Quantification of freezing behavior in a neutral Context B 30 min after injection of 3 mg/kg CNO (n = 8 saline, n = 7 CNO mice, two-tailed t test, *P = 0.012). Data are mean ± SEM. Panel a was created using Biorender.com.
Extended Data Fig. 12 Gating strategy for cell sorting.
Example of the gating strategy used for sorting GFP+ tdTomato+ (learning-associated) and GFP+ tdTomato- (non-learning-associated) astrocytes.
Extended Data Fig. 13 Validation of increased NFIA in learning-associated astrocytes.
a. Schematic of genetic system. b. Timeline for labeling learning-associated astrocytes. c. Representative immunostaining for NFIA. Arrow indicates tdTomato+ learning-associated astrocyte, arrowheads indicate tdTomato- astrocytes. d. Quantification of NFIA fluorescence intensity in tdTomato+ and tdTomato- astrocytes (n = 62 CA1 tdTomato- astrocytes, n = 61 CA1 tdTomato+ astrocytes, n = 48 DG tdTomato- astrocytes, n = 52 tdTomato+ DG astrocytes, n = 4 mice, nested t tests, *P = 0.021, ***P = 0.001). e. RT-qPCR quantification of neuronal Tuj1 mRNA in sorted cell populations. Tuj1 was de-enriched in GFP+ samples (n = 3 technical replicates, n = 3 biological replicates per group, one-way ANOVA and Tukey’s post hoc tests, ****P < 0.0001). f. RT-qPCR quantification of astrocytic Sox9 mRNA in sorted cell populations. Sox9 was enriched in GFP+ samples (n = 3 technical replicates, n = 3 biological replicates per group, one-way ANOVA and Tukey’s post hoc tests, **P = 0.002, ****P < 0.0001). g. RT-qPCR quantification of Nfia mRNA in sorted cell populations. Nfia was enriched in GFP+ tdTomato+ samples (n = 3 technical replicates, n = 3 biological replicates per group, one-way ANOVA and Tukey’s post hoc tests, *P = 0.022 (top), 0.031 (bottom). Data are mean ± SEM. Panel a was created using Biorender.com.
Extended Data Fig. 14 Fos deletion in learning-associated astrocytes impairs memory recall.
a. Timeline for Fos knockout in learning-associated astrocytes and examining memory recall. b. Schematic of genetic system. c. Confirmation of lack of Fos expression in dTomato+ learning-associated astrocytes relative to dTomato- astrocytes (N = 5 mice, two-tailed Welch’s t test, ****P < 0.0001). d. Quantification of freezing behavior during recall test in Context A. N = 10 Control (vehicle injected), N = 11 Fos cKO mice; two-tailed t test, *P = 0.014. e. Representative image showing lack of c-Fos expression in dTomato+ astrocytes. Representative of 5 mice. f. Low magnification image of learning-associated astrocyte labeling in CA1 and DG. Note that no dTomato+ astrocytes express c-Fos. Representative of 5 mice. Panel a was created using Biorender.com.
Supplementary information
Supplementary Table 1
DEGs and gene ontology from RNA-seq data of LAAs and non-learning associated astrocytes.
Source data
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Williamson, M.R., Kwon, W., Woo, J. et al. Learning-associated astrocyte ensembles regulate memory recall. Nature 637, 478–486 (2025). https://doi.org/10.1038/s41586-024-08170-w
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/s41586-024-08170-w
This article is cited by
-
Immediate-early genes Arc and c-Fos show divergent brain-wide expression following contextual fear conditioning
Communications Biology (2025)
-
Astrocytic Ca2+ prevents synaptic depotentiation by limiting repetitive activity in dendrites during motor learning
Nature Neuroscience (2025)
-
Astrocyte-Neuron Metabolic Synergies in Neurological Homeostasis and Disease
Neurochemical Research (2025)
-
The Power of Neuroglia in Driving Brain Function
Neurochemical Research (2025)
-
The Astrocyte: A New Component of The Engram Regulates Memory Recall
Neuroscience Bulletin (2025)