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Calcium dynamics in astrocyte processes during neurovascular coupling

Nature Neuroscience volume 18, pages 210–218 (2015)Cite this article

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Abstract

Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca2+) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca2+ signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca2+ sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca2+ increases in astrocyte processes but not in somata. These Ca2+ increases systematically precede the onset of functional hyperemia by 1–2 s, reestablishing astrocytes as potential regulators of neurovascular coupling.

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Figure 1: Somatic Ca2+ signals mediated by group 1 mGluRs in astrocytes and juxtaglomerular neurons of Aldh1l1-eGFP mice.
Figure 2: Synaptic activation of Ca2+ signals in glomerular astrocyte somata.
Figure 3: TBOA lowers the threshold for astrocyte responses but changes the basal state of the glomerular network.
Figure 4: Activation of Ca2+ signals in astrocytes by OSN stimulation requires post-synaptic activation of JG neurons and is differently modulated by group1 mGluRs.
Figure 5: GCaMP3 expression in glomerular layer astrocytes of adult Cx30-CreERT2; R26-lsl-GCaMP3 mice.
Figure 6: Effects of ATP and mGluR5 agonists on glomerular astrocytes from adult Cx30-CreERT2; R26-lsl-GCaMP3 mice.
Figure 7: Astrocyte processes in Cx30-CreERT2; R26-lsl-GCaMP3 mice reliably and rapidly respond to odor stimulation well before the onset of functional hyperemia.
Figure 8: Astrocyte processes respond well before the onset of functional hyperemia.

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Acknowledgements

We would like to thank C. Pouzat for his critical comments. Support was provided by INSERM, the Agence Nationale de la Recherche (Project Angioneurins R11036KK), the Leducq Foundation, the Human Frontier Science Program Organization (RGP008912009-C), the Fondation pour la Recherche Médicale (equipe FRM) and the US National Institutes of Health (MH084020 to D.E.B.). K.C. was supported by the Agence Nationale de la Recherche and the ERC Advanced Grant “Imaging-in-the-magnet.” D.G.L. was supported by a doctoral fellowship from École des Neurosciences de Paris (Paris School of Neuroscience) and a bursary from the Fondation pour la Recherche Médicale (FDT20130928252). The team of S.C. is part of the École des Neurosciences de Paris Ile-de-France network. We thank W. Fröstle (Ciba-Geigy) for the kind gift of CGP55845A (3-[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2(S)hydroxypropyl-P-benzyl-phosphonic acid).

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  1. Yo Otsu and Kiri Couchman: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale (INSERM), U1128, Paris, France

    Yo Otsu, Kiri Couchman, Declan G Lyons & Serge Charpak

  2. Laboratory of Neurophysiology and New Microscopies, Université Paris Descartes, Paris, France

    Yo Otsu, Kiri Couchman, Declan G Lyons & Serge Charpak

  3. Centre National de la Recherche Scientifique (CNRS), UMR 7203, Paris, France

    Mayeul Collot & Jean-Maurice Mallet

  4. Laboratory of Biomolecules, Université Pierre et Marie Curie, Paris, France

    Mayeul Collot & Jean-Maurice Mallet

  5. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Amit Agarwal & Dwight E Bergles

  6. CNRS UPR 3212, University of Strasbourg, Institute of Cellular and Integrative Neurosciences (INCI), Strasbourg, France

    Frank W Pfrieger

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Contributions

Y.O., K.C. and D.G.L. conducted the experiments, M.C. synthesized CaRuby-Nano, Y.O., K.C. and S.C. analyzed the data, Y.O., K.C., S.C. and J.-M.M. designed the research, A.A. and D.E.B. provided the R26-lsl-GCaMP3 mouse line, and F.W.P. provided the Cx30-CreERT2 mouse line. All authors edited the paper.

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Correspondence to Serge Charpak.

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Integrated supplementary information

Supplementary Figure 1 Amplitude and onset of astrocytic Ca2+ increases are dependent on stimulus duration.

(a) Ca2+ responses to trains of OSN stimuli (0.5, 1 or 2 s) in 2 astrocytes (a1-2) and 2 neurons (n1-2). The shaded area indicates stimulus duration (each pulse 800 µA, 100 µs). (b-c) Summary bar graphs (n = 9 cells, 4 mice) showing amplitude and latency of OSN-induced Ca2+ transients in astrocytes induced with 20 Hz stimulus trains of various durations (700 - 800 µA, 100 µs). Lines connect data from individual cells (open circles).

Supplementary Figure 2 Proposed schema summarizing results of slice recordings from juvenile mice as shown in Figures 1–4.

Black arrows: Electrical stimulation of OSNs (Stim) releases glutamate from OSN terminals, which activate glomerular dendrites via AMPAR, NMDAR, and mGluRs (the presence of particular mGluRs on neurons is dependent on the cell subtype), triggering dendritic release of glutamate, which in turn causes calcium increases in astrocyte somata via the action of mGluR5. Grey arrows: the puff application of t-ACPD directly activates mGluRs on both neurons and astrocytes.

Supplementary Figure 3 The expression of GCaMP3 in GLAST-CreERT2; R26-lsl-GCaMP3 mice is not specific for astrocytes in the adult olfactory bulb.

Horizontal sections of olfactory bulb from 3 month old uninjected (n = 2 mice, without the injection of 4-OHT) mice labeled for neurons (NeuN, left), the expression of GCaMP3 under the control of the GLAST promoter (GFP, left middle), and the astrocytic marker S100β (right middle). (a) In the EPL, GCaMP3-expressing granule cells and astrocytes (star) are visible. The dendrites of granule cells end at the base of JG neurons. Inset: spines are clearly visible on granule cell dendrites (scale bar 5 µm). Granule cells expressing GCaMP3 express the neuronal marker NeuN (arrowheads). (b) Astrocytes are present throughout the olfactory bulb, in the ONL where they enwrap blood vessels (star), and in the GL and EPL. Note the differing morphology of astrocytes in these three layers. (c) JG neurons (arrowhead) also express GCaMP3. In the lower half of this image, spiny granule cell dendrites can be seen extending into the bottom of the GL. ONL: olfactory nerve layer; GL: glomerular layer; EPL: external plexiform layer; MC/GCL: mitral cell/granule cell layer. Scale bar 50 µm. All images are standard deviation projections of confocal image stacks.

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Otsu, Y., Couchman, K., Lyons, D. et al. Calcium dynamics in astrocyte processes during neurovascular coupling. Nat Neurosci 18, 210–218 (2015). https://doi.org/10.1038/nn.3906

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