Extended Data Fig. 4: Sustained activation of both endogenous α1 and exogenous hM3Dq receptors results in the reduction of Ca²⁺ transients during motor running and the exhaustion of ER Ca²⁺ storage in astrocyte network.

(a) Images representing 4 independent experiments of running-induced astrocytic Ca²⁺ before and after 12 min of α1 agonist PE treatment. (b, c) The average amplitude of running-induced astrocytic Ca²⁺ activity before and after 12 min of PE treatment in the somas (b, n = 191, 210 respectively) and processes (c, n = 1190, 1232 respectively). Two sided Mann-Whitney test. All P < 0.001. Boxes show the first quartile, median and third quartile of the stack data. Whiskers show minima and maxima without outliers. (d) Schematic of the experimental design to sequentially activate endogenous (via PE) and exogenous Gq receptors (via CNO) in astrocytes expressing hM3Dq mcherry (asGq) and cytosolic GCaMP6f. (e) Images (up) and average amplitude (bottom) of astrocytic Ca²⁺ after CNO injection following 30 min post-PE treatment (1. CNO 1 min), and after PE application following 30 min post-CNO (2. PE 1 min) in both somas (left, n = 101, 110) and processes (right, n = 612, 1044) of astrocytes. Two sided Mann-Whitney test. P = 0.2903, 0.6547 for somas and processes respectively. (f) Images (up) of layer 1 astrocytes expressing hM3Dq (asGq-DREADD) and ER GCaMP sensors after saline injection, 30 min after PE bath application, and 1-5 min after CNO injection following 30 min of PE treatment. (bottom) The average amplitude of running-induced astrocytic ER Ca²⁺ changes after saline injection (control), 30 min after PE bath application, and 1-5 min after CNO injection following 30 min of PE treatment in the somas (n = 99, 101, 75 respectively) and processes (n = 2555, 3796, 2228 respectively). Two sided Mann-Whitney test. All P < 0.001. (g) Images (up) of astrocytes expressing hM3Dq (asGq-DREADD) and ER GCaMP sensors 10-15 min after saline injection, 30 min after CNO injection, and 1-5 min PE treatment following 30 min post-CNO injection. (bottom) The average amplitude of running-induced astrocytic ER Ca²⁺ changes after saline injection (control), 30 min after CNO injection, and 1–5 min of PE treatment following 30 min post-CNO injection in the somas (n = 330, 330, 179 respectively) and processes (n = 7631, 7631, 3785 respectively). These data indicate that both PE and asGq-DREADD activation eventually leads to the exhaustion of Ca²⁺ storage in the astrocyte ER, rendering astrocytes non-responsive to motor training. Two sided Mann-Whitney test. All P < 0.001. Data are mean ± s.e.m. ns P > 0.05, ***P < 0.001. Scale bar, 20 μm.

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