Abstract
Glutamate is the main excitatory neurotransmitter in the brain, acting through ionotropic receptors such as AMPA receptors (AMPARs). While their neuronal role is well established, AMPAR function in astrocytes remains poorly understood, especially in the hippocampus. In this study, we investigated the expression and functional properties of astrocytic AMPARs, focusing on their capacity to mediate calcium signaling and inter-cellular communication. Using astrocyte primary cultures, we confirmed the expression of GluA1 and GluA2 subunits, along with detectable levels of GluA4. Calcium imaging revealed that pharmacological AMPAR stimulation elicited intracellular Ca²⁺ signals in a subset of astrocytes which were markedly potentiated when desensitization was impaired. Notably, some of these responses persisted without extracellular Na⁺, compatible with the presence of functional calcium-permeable AMPARs (CP-AMPARs) in hippocampal astrocytes. However, sodium was required for full amplification of the Ca²⁺ signals, suggesting a synergistic role of Na⁺ and Ca²⁺ influx. Pharmacological inhibition of GluA2-lacking AMPARs reduced the responses, supporting the coexistence of GluA2-lacking and GluA2-containing subtypes. Sniffer-calcium assays suggested that strong AMPAR stimulation can engage ATP-dependent signaling, facilitating calcium wave propagation. Altogether, our results reveal functional and heterogeneous AMPAR populations in hippocampal astrocytes. Further investigations will elucidate their physiological roles, localization, and contribution to neuroglial interactions under physiological and pathological conditions.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Abbreviations
- AMPA:
-
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
- AMPAR:
-
AMPA receptor
- CP-AMPAR:
-
Calcium-permeable AMPA receptor
- CI-AMPAR:
-
Calcium-impermeable AMPA receptor
- CTZ:
-
Cyclothiazide
- DIV:
-
Days in vitro
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- FBS:
-
Fetal Bovine Serum
- GFAP:
-
Glial fibrillary acidic protein
- GLAST:
-
Glutamate aspartate transporter
- GFP:
-
Green fluorescent protein
- GRIA:
-
Glutamate receptor, ionotropic, AMPA
- HBSS:
-
Hank’s Balanced Salt Solution
- IP3R:
-
Inositol-1,4,5-triphosphate receptor
- LTP:
-
Long-term potentiation
- LTD:
-
Long-term depression
- NASPM:
-
1-Naphthyl acetyl spermine
- NBQX:
-
2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline
- PAM:
-
Positive allosteric modulator
- PBS:
-
Phosphate-buffered saline
- PBS-CM:
-
PBS with Calcium and Magnesium
- SEM:
-
Standard error of the mean
- TARP:
-
Transmembrane AMPA receptor regulatory protein
- TBS-T:
-
Tris-buffered saline with Tween-20
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Acknowledgements
We would like to sincerely thank Dr. Mercè Izquierdo for her invaluable support and guidance with the calcium imaging analysis. We are also grateful to Ms. Alba Pereda and Dr. Nadia Di Franco from the Histology Unit of the Institute of Neurosciences at the University of Barcelona for their assistance. We thank the Scientific and Technological Centers (CCiTUB), Universitat de Barcelona, and staff members Gemma Martin and Maria Calvo for their support and advice on fluorescence microscopy techniques. This work was supported by Grant PID2020-119932GB-I00 to D.S and PID2024-155505OB-I00 funded by MICIU/AEI/10.13039/501100011033 (MINECO) to D.S. and E.G.B; Grants PID2020-119305RB-I00 and PID2023-148439OB-I00 funded by MCIN/AEI/10.13039/501100011033 (MINECO) to X.G.; 2021SGR00292 from the Generalitat de Catalunya (to D.S. and X.G.); María de Maeztu Unit of Excellence CEX2021-001159-M, supported by MCIN/AEI/10.13039/501100011033, to the Institut de Neurociències, Universitat de Barcelona; PID2020–115823-GBI00 (MCIN/AEI/10.13039/501100011033) to L.J.D. and J.D.N.L.; PID2021-123732OB-I00 (MCIN/AEI/10.13039/501100011033) to S.G.; and the Spanish Ministry of Universities FPU fellowship FPU22/02071 to J.P.
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J.P. performed most of the experiments including calcium imaging, wester blotting and sniffer assays, analyzed the data, and wrote the initial draft of the manuscript. A.C. contributed to experimental design, performed supporting calcium imaging experiments, assisted in data analysis, figure preparation and original illustrations used to schematize the methodology. E.G.B. performed western blotting and immunofluorescence experiments, provided technical support, contributed to data interpretation, and assisted in manuscript preparation. R.G. contributed to experimental procedures and general laboratory support. S.G. contributed to the design and supervision of molecular biology experiments and provided key resources. X.G. provided scientific resources related to calcium imaging and contributed to the conceptual framework. He supervised aspects of data interpretation and revised the manuscript for intellectual content. L.J.D. and J.D.N.L. supported the project through institutional resources and supervision and offered conceptual input during the whole process. D.S. conceived and led the project, providing intellectual guidance throughout all stages. He secured funding, supervised the experimental design, data analysis, and interpretation, and was responsible for writing and critically revising the final version of the manuscript. All authors reviewed and approved the final version of the manuscript.
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Picañol, J., Castellanos, A., Gratacòs-Batlle, E. et al. Functional Ca2+-permeable AMPA receptors in hippocampal astrocytes mediate calcium signaling in vitro. Sci Rep (2026). https://doi.org/10.1038/s41598-026-41403-8
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DOI: https://doi.org/10.1038/s41598-026-41403-8