Fig. 5. ASD astrocytes decrease neuronal network firing and spine density in vitro.

a–l Primary hippocampal neurons dissociated from E16-18 WT mouse brains were co-cultured with human astrocytes isolated from CTRL or ASD organoids to measure spontaneous network activity with MEA. CTRL cultures refer to co-culturing of CTRL human astrocytes with hippocampal neuronal cultures (naturally containing some mouse astrocytes), and ASD cultures refer to co-culturing of ASD human astrocytes with hippocampal neuronal cultures (naturally containing some mouse astrocytes). “None” cultures refer to hippocampal neuronal cultures (containing some mouse astrocytes) without any addition of human astrocytes. a Representative images of co-cultures plated in a single well of a 48-well array plate. b ASD astrocyte co-cultures displayed decreased mean network firing rate when compared to CTRL co-cultures and None. (ANOVA with Tukey’s posthoc test None vs. CTRL p value = 0.98, None vs. ASD p value = 0.02, CTRL vs. ASD p value = 0.002, see also Supplementary Video 2). c ASD astrocytes also disrupted network synchronization when compared to CTRL astrocytes and None (None vs. CTRL p value = 0.65, None vs. ASD p value <0.0001, CTRL vs. ASD p value < 0.0001). d–f Representative raw traces of spontaneous spiking activity over a 10-s period. g ASD astrocytes decreased the # of network bursts when compared to None (None vs. ASD p value = 0.02, CTRL vs. ASD p value = 0.06). h, i ASD astrocytes did not affect average burst duration but decreased the # of spikes per burst network (None vs. ASD p value = 0.005, CTRL vs. ASD p value <0.0001). j–l Representative raster plots (4-min) demonstrated the decrease in burst number and spikes per bursts in the ASD co-culture group compared to CTRL co-culture and None. m–o Spine density quantified in a 10 μm dendritic segment at least 20 μm away from the soma in hippocampal neurons co-cultured with ASD or CTRL human astrocytes. m Immunostained co-cultures with human astrocytes infected with CMV GFP lentivirus (pseudo colored red) prior to co-culture. n Neurons were labeled with a αCamKII GFP AAV at DIV5 and fixed at DIV18 (10 μm dendritic segments shown at bottom). o ASD astrocytes decreased spine density on primary hippocampal neurons (None vs. CTRL p value = 0.78, None vs. ASD 0.009, CTRL vs. ASD p value = 0.004). These results provide direct evidence that ASD astrocytes influence structural and functional properties of neurons that weaken electrophysiological activity. Scale bar = 100 μm. Data are represented as mean ± SEM. MEA: None n = 6 wells, CTRL n = 18 wells, 3 distinct lines; ASD n = 22–24 wells, 4 distinct lines. Spine quantification: None n = 25 neurons, CTRL n = 71 neurons co-cultured with 4 distinct lines, ASD n = 81 neurons co-cultured with 5 distinct lines. CTRL: Co-cultures of CTRL human astrocytes with mouse hippocampal neurons. ASD: Co-cultures of ASD human astrocytes with mouse hippocampal neurons. None: Mouse hippocampal neuron cultures with no human astrocytes.