Structural basis of astrocytic Ca2+ signals at tripartite synapses

Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.

(e) A comparison of width measurements between proximal and distal regions. The anatomical organization of nodes and connecting shafts was equally present in proximal and distal regions (n = 54 for proximal shaft, n = 90 for distal shaft, n = 42 for proximal node, n = 111 for distal node, from 7 slices; Mann-Whitney U test, p (two-tailed) = 0.053 for shafts and p (two-tailed) = 0.70 for nodes, N.S.: not significant). Data are presented as median, interquartile range, and whiskers 10-90%.
(f) A comparison of node density between proximal and distal regions, showing that they are distributed at similar density (n = 13 cells from 7 slices; Mann Whitney U-test p (two-tailed) = 0.30, N.S.: not significant). Data are presented as median, interquartile range, and whiskers 10-90%. (b) Image of spines with one node (left) and two nodes (right).

Single node
(c) Percentage of spines with one or two nodes. The majority of spines have one node.
(d) Comparison of spine head width between spines with one and two nodes. Spines with two nodes have wider spine heads (n = 92 for double node spine, n = 11 for single node spine from 22 slices; Mann Whitney U test, **** p (twotailed) < 0.0001). Data are presented as median, interquartile range, and whiskers 10-90%.
Supplementary Figure 3  (c-e) Comparison of amplitude (c), duration (d), and spread (e) of spontaneous Ca 2+ events between major processes and spongiform domain. In the major astrocytic branches, Ca 2+ transients had greater amplitudes and were larger in space (n = 60 events for major processes, n = 1583 events for spongiform structure, from 5 slices; Mann-Whitney U test, ****p (two-tailed) < 0.0001, N.S.: Not Significant p (two-tailed) = 0.820).

Non-confined
Supplementary Figure 5 Supplementary Figure 5｜Nodes are a prominent site for Ca 2+ signals in astrocytes (a) Comparison of GCaMP6s-labelled node size (area) and ZsGreen-labelled node size (n = 52 nodes from 22 slices for GCaMP6s and n = 103 nodes from 21 slices for ZsGreen; Mann Whitney U test, N.S.: Not Significant p (two-tailed) = 0.50). Data are presented as median, interquartile range, and whiskers 10-90%.
(c) Percentage of nodes that form branch points. Similar to ZsGreen-labelled nodes, the majority of GCaMP6s-labelled nodes formed branch points (n = 52 nodes from 22 slices).
(d) Time-lapse images of spontaneous Ca 2+ event acquired at 8 Hz and corresponding STED image of astrocytic process.
(e) Percentage of Ca 2+ events that were confined to single nodes, non-confined and that occurred at other undefined structures.
(f) Ca 2+ traces of the Ca 2+ propagation event (right) from ROIs indicated on the corresponding STED image (left). N 1 : node that initiated the Ca 2+ event, S: neighboring shaft, N 2 : connected neighboring node (g) Percentage of Ca 2+ events that were initiated at nodes and those that were initiated at nodes and/or shafts, among the non-confined events.