Neuroscience

A spatial threshold for astrocyte calcium surge

Justin Lines author has email address
Andres Baraibar
Carmen Nanclares
Eduardo D Martín
Juan Aguilar
Paulo Kofuji
Marta Navarrete
Alfonso Araque

Department of Neuroscience, University of Minnesota, Minneapolis, USA
Instituto Cajal, CSIC, Madrid, Spain
Experimental Neurophysiology Group, Hospital Nacional de Parapléjicos SESCAM, Toledo, Spain

https://doi.org/10.7554/eLife.90046.2

Open access
Copyright information

Figures and data

Imaging astrocyte structure and function simultaneously in vivo.
(A), Scheme of in vivo preparation to image astrocyte Ca²⁺ and structure. (B), SR101-stained astrocyte structure, GCaMP6 to monitor astrocyte Ca²⁺ signal, and merge. Scale bar = 50 µm. (C), Regions of interest (ROIs) from SR101-stained structure of somas (blue) and arborizations (red). (D), Ca²⁺ traces from B from somas (blue) and arborizations (red). Scale = F/F_o, 10 s. (E), SR101-stained astrocyte (left), ROIs outlining soma and arborization (center) and ROIs defining the soma and domains (right). Scale bar = 10 µm. (F), Pseudocolor Ca²⁺ image during basal (left) and hindpaw electrical stimulation (right). (G), Ca²⁺ traces from f from domains (salmon), arborization (red) and soma (blue). Scale = F/F_o, 10 s.

Population arborization calcium is correlated to population soma activity.
(A), SR101 staining. Scale bar = 50 µm. (B), pseudocolor Ca²+ images at basal and stimulation. (C), ROIs of soma and arborizations/domains along with activity during stimulation. (D), Proportion of subcellular responses to stimulation. (E), Percentage of active arborizations vs. percent of somas active. (F), Percentage of domains active vs. percent of somas active. Mean ± SEM. Pearson correlation.

Astrocyte calcium responses originate in the arborization before the soma.
(A), Astrocyte with regions of interest (ROIs). Scale bar = 10 µm. (B), Pseudocolor Ca²⁺ image. (C), Ca²⁺ traces in B from domains (pink), arborization (red), and the soma (blue). Scale = F/F_o, 5 s. (D), Raster plot of astrocyte somas (blue) and arbors (red) in response to stimulation (gray). (E), Average calcium traces from somas (blue) and arborizations (red) aligned to their respective soma onset. (F), Soma and arbor latency to response (left), event rise time (center) and event decay time (right). Mean ± SEM. ‘**’ ≡ p < 0.01 and ‘***’ ≡ p < 0.001 using paired student t-test.

A spatial threshold for astrocyte calcium in the soma to reach astrocyte calcium surge.
(A), Astrocyte and ROIs. Scale bar = 10 µm. (B), Pseudocolor Ca²⁺ images during basal and different frequency of stimulations. (C), Scheme of domains (red) and soma (blue) Ca²⁺ activity from B. (D), Percentage of active domains vs. stimulus duration, intensity and frequency. (E), Active state of soma for individual astrocytes vs. percentage of active domains (red). Data were fit to a Heaviside step function (blue dotted line). (F), Percentage of active domains necessary to elicit soma activation vs. stimulus duration, intensity and frequency. Blue dotted lines denote 22.6% spatial threshold. (G), Soma fluorescence versus percentage of active domains (red). Data were fit to a sigmoidal function (blue) and a blue dotted line denotes 22.6% spatial threshold. (H), Percentage of active domains in the absence of soma activation versus active domains before and after soma activation. Blue line denotes 22.6% spatial threshold. (I), Schematic showing subthreshold and suprathreshold astrocyte calcium activity. Mean ± SEM. ‘***’ ≡ p < 0.001 and ‘ns’ ≡ p > 0.05 using 1-way ANOVA or student t-test.

The spatiotemporal properties of astrocyte calcium surge.
(A), The time course of calcium surge following sensory stimulation. The average of 10 sequential frames (2 s) ending on the time from stimulation onset in seconds. (B), Temporal Map of domain temporal order relative to soma activation from A. (C), Average distance between pairs of active domains in the absence of soma activation versus before and after soma activation (D), Average time between pairs of active domains in the absence of soma activation versus before and after soma activation. Mean ± SEM. (E), Comparison between activated domain onset relative to soma activation vs. radius from the center of the soma. ‘***’ ≡ p < 0.001 using paired and unpaired student t-test. Pearson correlation.

The spatial activation of domain Ca²⁺ remains below the spatial threshold in mice lacking the IP₃ receptor Type-2.
(A), SR101 staining. Scale bar = 50 µm. (B), Pseudocolor Ca²⁺ images at basal and stimulation. (C), Traces from astrocytes in B. Scale = F/F_o, 10 s. (D), Percentage of domains (left) arborizations (center) and somas (left) active at basal (open) and stimulation (hashed) in IP₃R2^-/- mice. (E), Percentage of domains active in wildtype (filled) and IP₃R2^-/- mice (hashed). Blue line denotes 22.6% spatial threshold. (F), Probability of soma activation in wildtype (filled) and IP₃R2^-/- mice (hashed). Mean ± SEM. ‘***’ ≡ p < 0.001 using paired and unpaired student t-test.

Increases in slow-inward currents occurs with astrocyte calcium surge.
(A), Scheme of cortical brain slice experiments to image astrocyte Ca²⁺ and record slow-inward currents (SICs) with ATP application. (B), Example traces of a miniature excitatory post synaptic current (mEPCS) and a slow inward current (SIC) (upper) and SICs following ATP puff (black bar) (lower). (C), Pseudocolor Ca²⁺ images at basal and ATP with traces of responses to puff (black bar) in the soma (blue), arbor (red), and domains (salmon). Scale bar = 10 µm. Scale = F/F_o, 10 s. (D), Active state of soma for individual astrocytes vs. percentage of active domains (red), with fit to a Heaviside step function (blue line). (E), Percentage of domains active in response to ATP puff. Blue dotted line denotes spatial threshold from D. (F), SIC frequency in response to ATP puff. (G), Pearson correlation between percent active domains vs. SIC frequency. Blue dotted line denotes spatial threshold from D. Mean ± SEM. ‘***’ ≡ p < 0.001 using 1-way ANOVA and t-test of Pearson correlation.

Semi-automatic method for the segmentation of astrocyte morphology.
(A), SR101 stained astrocyte population with an outlined cell. Scale bar = 50 µm. (B), Selected astrocyte placed in polar coordinates with rings overlaid to assess structural fluorescence. Scale bar = 10 µm. (C), Average fluorescence of rings centered on astrocyte soma as radius is extended outward. Note, overlay of values from rings in B. (D), Fluorescence of rings in panel B as a function of angle. (E), Regions of Interest (ROIs) from algorithm. (F), Calcium pseudocolor image during basal and stimulation. (G), Calcium traces from f during stimulation. Scale = F/F_o, 10 s.

Distinct dynamics of domains before and after soma onset.
(A), Average calcium traces from somas (blue) and domains activating before the soma (pre-soma; green) and after the soma (post-soma; pink) aligned to their respective soma onset. (B), Pre-soma and post-soma latency to response relative to their respective soma onset. (C), Event rise time. (D), Event decay time. Mean ± SEM. ‘***’ ≡ p < 0.001 using paired student t-test.

Sign up for email alerts