Fig. 7. ASD astrocytes with modulated Ca²⁺ signaling do not induce impaired network activity and memory.

a–e Human astrocytes were infected with either the non-targeting shRNA lentivirus (Non) or the IP₃Rs shRNA lentivirus (KD) and co-cultured with primary hippocampal neurons. a Decrease in spiking pattern in untreated ASD Non co-cultures was corrected upon IP₃Rs KD in ASD astrocytes. b As in Fig. 5, ASD co-cultures displayed decreased mean network firing rate when compared to CTRL co-cultures. Modulation of ASD astrocyte Ca²⁺ mobilization conferred protection against deficits in mean firing rate (see also Supplementary Video 3). c Similarly, ASD Non co-cultures displayed lower synchronicity that was corrected in ASD KD co-cultures. d Downregulation of IP₃R rescued the phenotypes in network burst number (red hash marks surrounded by red boxes) and spikes per bursts (red hash marks) in the ASD astrocytes. e Unlike untreated ASD astrocytes, ASD KD astrocytes displayed similar number of network bursts when compared to CTRL astrocytes. These findings suggest that fine-tuning ASD astrocyte Ca²⁺ levels protects against deficits induced by untreated ASD astrocytes. f–h CTRL Non astrocytes, ASD Non astrocytes, and ASD KD astrocytes were transplanted into the brains of neonatal Rag2^KO mice. f Similar to the previous experiment, there was no significant difference in the rate of acquisition learning between CTRL and ASD mice. g As in Fig. 4, ASD Non chimeric mice showed reduced freezing behavior when exposed to the fear context relative to CTRL Non chimeric mice. However, this difference was eliminated between ASD KD and CTRL Non chimeric mice, indicating that amelioration of cytosolic Ca²⁺ in ASD astrocytes prevents fear memory deficits. h No significant differences were detected in freezing behavior between CTRL, ASD, and ASD KD mice during cue presentation in a novel context. Together, these results indicate that exaggerated Ca²⁺ release from internal stores in ASD astrocytes is responsible for neuronal network activity and memory deficits caused by these cells. MEA: CTRL Non co-cultures n = 12 wells, 3 distinct lines; ASD Non n = 10–16 wells, 4 distinct lines; ASD KD n = 15–16 wells, 4 distinct lines. Fear testing: CTRL Non n = 17 male and female mice, transplanted with 2 distinct lines, ASD Non n = 22 male mice, transplanted with 4 distinct lines, ASD KD n = 24 male mice, transplanted with 4 distinct lines. CTRL Non: Control human astrocytes infected with non-targeting shRNA lentivirus. ASD Non: ASD astrocytes infected with non-targeting shRNA lentivirus. ASD KD: ASD astrocytes infected with IP₃R shRNA lentivirus.