Fig. 4

Factors released by 22L-derived reactive astrocytes impairs synapse integrity and spine density. a Representative images of primary cortical neurons treated with CT-ACM or 22L-ACM for 72 h and co-immunostained for the pre- and post-synaptic markers synaptophysin (SYP, green) and PSD-95 (red), respectively, and MAP2 (blue). Arrows point at puncta of co-localization of synaptophysin and PSD-95. b Quantification of co-localized puncta in CT-ACM- and 22L-ACM-treated primary neurons. c Representative Western blots and densitometric analysis of synaptophysin (SYP) and PSD-95 expression normalized per expression of β-actin in neuronal cultures treated with CT-ACM or 22L-ACM. d Representative images of neuronal cultures treated with CT-ACM or 22L-ACM for 72 h and co-immunostained for a spine marker Drebrin (red) and MAP2 (green). Arrows point at the secondary dendritic spine branches. Quantification of spine size and density in primary neurons incubated with CT-ACM or 22L-ACM. In b–d, data represent mean ± SE, n = 3 independent experiments in which CT-ACM or 22L-ACM was collected from cultures established form individual animals, 50 neurons (in b) and 40 neurons (in d) were counted for each condition. *p < 0.05 (two tailed, unpaired student t test with Welch's correction). Scale bar = 25 µm for a and 10 µm for d panels