Control of Ca2+ signals by astrocyte nanoscale morphology at tripartite synapses

doi:10.1002/glia.24258

. 2022 Dec;70(12):2378-2391.

doi: 10.1002/glia.24258. Epub 2022 Sep 13.

Control of Ca²⁺ signals by astrocyte nanoscale morphology at tripartite synapses

Audrey Denizot¹, Misa Arizono^{2

3

4}, U Valentin Nägerl^{2

3}, Hugues Berry^{5

6}, Erik De Schutter¹

Affiliations

¹ Computational Neuroscience Unit, Okinawa Institute of Science and Technology, Onna-Son, Japan.
² Interdisciplinary Institute for Neuroscience, Université de Bordeaux, Bordeaux, France.
³ Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France.
⁴ Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
⁵ LIRIS, UMR5205 CNRS, Univ Lyon, Villeurbanne, France.
⁶ INRIA, Villeurbanne, France.

PMID: 36097958
PMCID: PMC9825906
DOI: 10.1002/glia.24258

Control of Ca²⁺ signals by astrocyte nanoscale morphology at tripartite synapses

Audrey Denizot et al. Glia. 2022 Dec.

. 2022 Dec;70(12):2378-2391.

doi: 10.1002/glia.24258. Epub 2022 Sep 13.

Authors

Audrey Denizot¹, Misa Arizono^{2

3

4}, U Valentin Nägerl^{2

3}, Hugues Berry^{5

6}, Erik De Schutter¹

Affiliations

¹ Computational Neuroscience Unit, Okinawa Institute of Science and Technology, Onna-Son, Japan.
² Interdisciplinary Institute for Neuroscience, Université de Bordeaux, Bordeaux, France.
³ Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France.
⁴ Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
⁵ LIRIS, UMR5205 CNRS, Univ Lyon, Villeurbanne, France.
⁶ INRIA, Villeurbanne, France.

PMID: 36097958
PMCID: PMC9825906
DOI: 10.1002/glia.24258

Abstract

Much of the Ca²⁺ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so-called spongiform domain. A growing body of literature indicates that those astrocytic Ca²⁺ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca²⁺ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super-resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca²⁺ activity. Simulations demonstrate that the nano-morphology of astrocytic processes powerfully shapes the spatio-temporal properties of Ca²⁺ signals and promotes local Ca²⁺ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron-astrocyte communication at so-called tripartite synapses, where astrocytic processes come into close contact with pre- and postsynaptic structures.

Keywords: calcium microdomains; computational neuroscience; intracellular signaling; nano-morphology; reaction-diffusion simulations.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

**FIGURE 1**
Geometries and kinetic scheme used for simulating ${Ca}^{2 +}$ dynamics in node/shaft structures of the gliapil. (a1) Representative STED image showing the astrocytic spongiform domain. Zoom‐in images show its anatomical units: Nodes and shafts. (a2) Distribution of the ratio between node width and the width of its neighboring shaft, measured using STED microscopy, n = 28. (b) Idealized geometries designed to reproduce the node/shaft ultrastructure of the spongiform domain. Nodes are approximated as spheres of diameter 380 nm and shafts as 1 μm‐long cylinders, based on their recent characterization in live tissue by Arizono et al. (2020) (Figure 1i–k. Shaft width varied based on experimentally‐observed node‐shaft width ratios reported in panel b: $d_{shaft} = d_{0} = 380$ nm, $d_{shaft} = \frac{d_{0}}{2}$ and $d_{shaft} = \frac{d_{0}}{3}$ . The geometries designed in this study, referred to as “5 nodes,” contain 5 identical nodes and 4 identical shafts. Unless specified otherwise, ER geometry (black) also consists in node/shaft successions (see Methods). The associated cytosolic volume, plasma and ER membrane areas are presented in Table 1. (c) Example of a ${Ca}^{2 +}$ trace of the model, recorded in one node. ${Ca}^{2 +}$ activity is quantified by measuring time to 1^st peak, peak probability, amplitude, duration and frequency. A peak is detected when the number of ${Ca}^{2 +}$ ions in the recorded node increases above peak threshold ${[Ca]}_{b} + n σ_{Ca}$ (see Methods). (d) Biochemical processes included in the model. ${Ca}^{2 +}$ can enter/exit the cytosol from/to the extracellular space or the endoplasmic reticulum (ER), resulting from the activity of ${Ca}^{2 +}$ channels/pumps. ${Ca}^{2 +}$ and IP₃ diffuse in the cytosol following Brownian motion. The kinetics of ${IP}_{3} R$ channels corresponds to the 8‐state Markov model from Denizot et al. (2019), adapted from De Young and Keizer (1992); Bezprozvanny et al. (1991). When both IP₃ and ${Ca}^{2 +}$ are bound to IP₃R activating binding sites, the IP₃R is in open state and ${Ca}^{2 +}$ enters the cytosol. ${Ca}^{2 +}$ can activate phospholipase C δ (PLCδ), which results in the production of IP₃. For more details, please refer to Denizot et al. (2019). Neuronal stimulation is simulated as an infusion of IP₃ in the cytosol and the opening of ${Ca}^{2 +}$ channels at the plasma membrane with an influx rate $k_{Ca}$ (see Methods).

**FIGURE 2**
Simulations confirm that thin shafts favor node compartmentalization. (a) Geometries of different shaft widths $d_{shaft}$ , $d_{shaft} = d_{0}$ , $\frac{d_{0}}{2}$ and $\frac{d_{0}}{3}$ , used in the bleaching simulations. Blue color represents the bleached volume, which varied depending on the value of $d_{shaft}$ in order to fit experimental values of $I_{0}$ and $I_{\inf}$ . (b) Representative experimental (top) and simulation (bottom) traces for different shaft width values. $I_{0}$ , $I_{\inf}$ and $τ$ were calculated using Equation (1). Note that simulations were also performed for $d_{shaft} = \frac{d_{0}}{3}$ . (c) Quantification of $I_{0}$ (c1), $I_{\inf}$ (c2), and $τ$ (c3) values in simulations (red) compared to experiments (black). Note that no experimental data was available for $d_{shaft} = \frac{d_{0}}{3}$ . In c1 and c2, n = 5 × 2 and 20 × 3 for experiments and simulations, respectively. Data are presented as mean ± STD. In c3, n = 66 and n = 20 × 3 for experiments and simulations, respectively. $τ$ is negatively correlated to $d_{shaft}$ in experiments (n = 66 from 7 slices; Spearman r = −0.72, p < 0.001***) and simulations (n = 60; Spearman r = −0.89, p < 0.001***). Black and red lines represent curve fit of $τ$ as a function of $d_{shaft}$ of the form $τ = a^{*} \frac{1}{d_{shaft}} + b$ for experiments and simulations, respectively. (d) Schematic summarizing the conclusion of this figure: Diffusion flux increases with $d_{shaft}$ . In that sense, thin shafts favor node compartmentalization. Data in panels c1 and c2 are represented as mean ± STD, n = 20 for each geometry.

**FIGURE 3**
${Ca}^{2 +}$ peak probability, amplitude and duration increase when shaft width decreases. (a) (top) Neuronal stimulation protocol simulated for each geometry: Node 1 was stimulated at t = t ₀ = 1 s, while ${Ca}^{2 +}$ activity was monitored in node 2. Representative ${Ca}^{2 +}$ traces for shaft width $d_{shaft} = d_{0}$ (red), $\frac{d_{0}}{2}$ (black) and $\frac{d_{0}}{3}$ (blue), expressed as SNR (see Methods). (b) Quantification of the effect of $d_{shaft}$ on ${Ca}^{2 +}$ signal characteristics data are represented as mean ± STD, n = 20. ${Ca}^{2 +}$ peak probability increases (***, b1), time to 1^st peak decreases (***, b2), peak amplitude (***, b3) and duration (***, b4) increase when $d_{shaft}$ decreases. (c) ${Ca}^{2 +}$ residency time in node 1 increases when $d_{shaft}$ decreases (***, n = 300). (d) Schematic summarizing the main result from this figure: ${Ca}^{2 +}$ peak probability and amplitude increase when shaft width decreases.

**FIGURE 4**
${Ca}^{2 +}$ imaging confirms that swelling attenuates local spontaneous ${Ca}^{2 +}$ activity. (a, b) (top) Confocal images of the astrocytic spongiform domain expressing GCaMP6s at baseline (basal, a) and in hypo‐osmotic condition (HOC, b), measured in organotypic hippocampal cultures (resolution: 200 nm in x–y, 600 nm in z). (bottom) representative traces of spontaneous ${Ca}^{2 +}$ events from ROIs indicated in (a) (a1–a3) and (b) (b1–b3). (c, d) ${Ca}^{2 +}$ peak amplitude (c) and duration (d) of spontaneous ${Ca}^{2 +}$ events are significantly smaller in hypo‐osmotic conditions (HOC) compared to basal conditions (Basal). (e, f) Amplitude (e) and duration (f) of spontaneous ${Ca}^{2 +}$ events do not significantly vary when measured twice in a row (1, 2) in the absence of HOC. Lines represent measurements in the same cell, before and after applying hypo‐osmotic stress.

**FIGURE 5**
Thin shafts favor a more robust signal propagation upon repeated neurotransmitter release events. (a) (top) Neuronal stimulation protocol: Node 1 is stimulated at t = t ₀ = 5 s, node 2 at $t_{0} + τ_{IP 3}$ , node 3 at $t_{0} + 2 τ_{IP 3}$ and node 4 at $t_{0} + 3 τ_{IP 3}$ , $k_{Ca}$ = 0 $s^{- 1}$ . ${Ca}^{2 +}$ activity is recorded in node 5. (bottom) representative ${Ca}^{2 +}$ traces in node 5 for shaft width $d_{shaft} = d_{0}$ (red), $\frac{d_{0}}{2}$ (black) and $\frac{d_{0}}{3}$ (blue), with $τ_{IP 3}$ = 250 ms (left) and 3000 ms (right), expressed as SNR (see Methods). For all values of $τ_{IP 3}$ tested, simulation time was 25 s. (b1) Time to 1^st peak increases with $τ_{IP 3}$ for $d_{shaft}$ = $d_{0}$ (***), $\frac{d_{0}}{2}$ (***) and $\frac{d_{0}}{3}$ (***). T‐tests revealed that for any value of $τ_{IP 3}$ , time to 1^st peak is higher for $d_{shaft}$ = $d_{0}$ compared to $d_{shaft}$ = $\frac{d_{0}}{2}$ and $\frac{d_{0}}{3}$ . Time to 1^st peak is significantly higher when $d_{shaft}$ = $\frac{d_{0}}{2}$ compared to $d_{shaft}$ = $\frac{d_{0}}{3}$ , for most values of $τ_{IP 3}$ (p = 0.032*, 0.0025**, 0.034*, 0.016*, and 0.019* for $τ_{IP 3}$ = 250, 500, 1000, 4000, and 5000 ms, respectively). (b2) ${Ca}^{2 +}$ peak probability in node 5 is lower for $d_{shaft}$ = $d_{0}$ compared to $d_{shaft}$ = $\frac{d_{0}}{2}$ and $\frac{d_{0}}{3}$ . ${Ca}^{2 +}$ peak probability decreases as $τ_{IP 3}$ increases for $d_{shaft}$ = $d_{0}$ (***). (c) ${Ca}^{2 +}$ peak probability in node 5 (colorbar) as a function of $τ_{IP 3}$ and of the probability of failure of node stimulation $p_{fail}$ , for $d_{shaft}$ = $d_{0}$ (c1), $d_{shaft}$ = $\frac{d_{0}}{2}$ (c2) and $d_{shaft}$ = $\frac{d_{0}}{3}$ (c3), with $p_{fail} \in [0, 1]$ . (d) Schematic summarizing the main conclusion of this figure: Decreased shaft width allows signal propagation despite omitted node stimulation, thus favoring more robust signal propagation. Data are represented as mean ± STD, n = 20 for each value of $d_{shaft}$ and of $τ_{IP 3}$ . Lines in panel B are guides for the eyes.

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References

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1. Ahmadpour, N. , Kantroo, M. , & Stobart, J. L. (2021). Extracellular calcium influx pathways in astrocyte calcium microdomain physiology. Biomolecules, 11(10), 1467. 10.3390/biom11101467 - DOI - PMC - PubMed
1. Arizono, M. , Bancelin, S. , Bethge, P. , Chéreau, R. , Idziak, A. , Inavalli, V. V. G. K. , Pfeiffer, T. , Tønnesen, J. , & Nägerl, U. V. (2021). Nanoscale imaging of the functional anatomy of the brain. Neuroforum, 27(2), 67–77. 10.1515/nf-2021-0004 - DOI
1. Arizono, M. , Inavalli, V. V. G. K. , Bancelin, S. , Fernández‐Monreal, M. , & Nägerl, U. V. (2021). Super‐resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge. Glia, 69(6), 1605–1613. 10.1002/glia.23995 - DOI - PubMed
1. Arizono, M. , Inavalli, V. V. G. K. , Panatier, A. , Pfeiffer, T. , Angibaud, J. , Levet, F. , Ter Veer, M. J. T. , Stobart, J. , Bellocchio, L. , Mikoshiba, K. , Marsicano, G. , Weber, B. , Oliet, S. H. R. , & Nägerl, U. V. (2020). Structural basis of astrocytic Ca2+ signals at tripartite synapses. Nature Communications, 11(1), 1–15. 10.1038/s41467-020-15648-4 - DOI - PMC - PubMed

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[1] Agarwal, A. , Wu, P.‐H. , Hughes, E. G. , Fukaya, M. , Tischfield, M. A. , Langseth, A. J. , Wirtz, D. , & Bergles, D. E. (2017, February). Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron, 93(3), 587–605.e7. 10.1016/j.neuron.2016.12.034 - DOI - PMC - PubMed

[2] Agarwal, A. , Wu, P.‐H. , Hughes, E. G. , Fukaya, M. , Tischfield, M. A. , Langseth, A. J. , Wirtz, D. , & Bergles, D. E. (2017, February). Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron, 93(3), 587–605.e7. 10.1016/j.neuron.2016.12.034 - DOI - PMC - PubMed

[3] Ahmadpour, N. , Kantroo, M. , & Stobart, J. L. (2021). Extracellular calcium influx pathways in astrocyte calcium microdomain physiology. Biomolecules, 11(10), 1467. 10.3390/biom11101467 - DOI - PMC - PubMed

[4] Ahmadpour, N. , Kantroo, M. , & Stobart, J. L. (2021). Extracellular calcium influx pathways in astrocyte calcium microdomain physiology. Biomolecules, 11(10), 1467. 10.3390/biom11101467 - DOI - PMC - PubMed

[5] Arizono, M. , Bancelin, S. , Bethge, P. , Chéreau, R. , Idziak, A. , Inavalli, V. V. G. K. , Pfeiffer, T. , Tønnesen, J. , & Nägerl, U. V. (2021). Nanoscale imaging of the functional anatomy of the brain. Neuroforum, 27(2), 67–77. 10.1515/nf-2021-0004 - DOI

[6] Arizono, M. , Bancelin, S. , Bethge, P. , Chéreau, R. , Idziak, A. , Inavalli, V. V. G. K. , Pfeiffer, T. , Tønnesen, J. , & Nägerl, U. V. (2021). Nanoscale imaging of the functional anatomy of the brain. Neuroforum, 27(2), 67–77. 10.1515/nf-2021-0004 - DOI

[7] Arizono, M. , Inavalli, V. V. G. K. , Bancelin, S. , Fernández‐Monreal, M. , & Nägerl, U. V. (2021). Super‐resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge. Glia, 69(6), 1605–1613. 10.1002/glia.23995 - DOI - PubMed

[8] Arizono, M. , Inavalli, V. V. G. K. , Bancelin, S. , Fernández‐Monreal, M. , & Nägerl, U. V. (2021). Super‐resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge. Glia, 69(6), 1605–1613. 10.1002/glia.23995 - DOI - PubMed

[9] Arizono, M. , Inavalli, V. V. G. K. , Panatier, A. , Pfeiffer, T. , Angibaud, J. , Levet, F. , Ter Veer, M. J. T. , Stobart, J. , Bellocchio, L. , Mikoshiba, K. , Marsicano, G. , Weber, B. , Oliet, S. H. R. , & Nägerl, U. V. (2020). Structural basis of astrocytic Ca2+ signals at tripartite synapses. Nature Communications, 11(1), 1–15. 10.1038/s41467-020-15648-4 - DOI - PMC - PubMed

[10] Arizono, M. , Inavalli, V. V. G. K. , Panatier, A. , Pfeiffer, T. , Angibaud, J. , Levet, F. , Ter Veer, M. J. T. , Stobart, J. , Bellocchio, L. , Mikoshiba, K. , Marsicano, G. , Weber, B. , Oliet, S. H. R. , & Nägerl, U. V. (2020). Structural basis of astrocytic Ca2+ signals at tripartite synapses. Nature Communications, 11(1), 1–15. 10.1038/s41467-020-15648-4 - DOI - PMC - PubMed

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Control of Ca²⁺ signals by astrocyte nanoscale morphology at tripartite synapses

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Control of Ca²⁺ signals by astrocyte nanoscale morphology at tripartite synapses

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