Fig. 6. Modulation of Ca²⁺ signaling in ASD astrocytes.

a, b ASD astrocytes were transduced with an shRNA lentivirus to downregulate IP₃Rs (KD). As a control, ASD astrocytes were infected with a non-targeting shRNA lentivirus (Non). c–f A high throughput Ca²⁺ mobilization assay was optimized (see “Methods”) for validation of Ca²⁺ modulation upon knockdown of IP₃Rs. c ASD KD astrocytes responded to the application of a cocktail of Gq activators (red line, 50 μM DHPG, 50 μM norepinephrine, 50 μM ATP, and 50 nM Endothelin 1) with diminished Ca²⁺ mobilization compared to ASD Non astrocytes. d ASD KD astrocytes did not display reduced Ca²⁺ mobilization in response to activation of Gs GPCR signal transduction (12.5 nM forskolin), which does not rely on IP₃R mediated Ca²⁺ release from the ER, indicating the specificity of the KD system. e No differences were detected in the total Ca²⁺ concentration stored in the ER between ASD KD or Non astrocytes. Application of thapsigargin (2 μM, red line) depleted the ER Ca²⁺ and inhibited reuptake by Sarco/ER Ca²⁺-ATPase (SERCA) Ca²⁺ pumps. f ASD KD astrocytes showed lower Ca²⁺ responses to Gq activation compared ASD astrocytes, which indicates that IP₃R reduction in ASD astrocytes modulated evoked increases in cytosolic Ca²⁺ from internal stores without changing its total concentration in the ER (Gq activation vs. Thapsigargin).