Fig 2: Deleterious effects of factors released by 22L-astrocytes on neuronal morphology, viability and expression of functional genes. a Analysis of expression of synaptogenic and neurotrophic genes in 22L-PACs normalized by the expression levels in CT-PACs using qRT-PCR. b Schematic illustration of experiments on treatment of mouse primary cortical neuronal cultures with astrocyte-conditioned media (ACM) collected from CT-PACs and 22L-PACs. c Representative images of primary neuronal cultures isolated from P1–P2 mice and co-immunostained for MAP2 (green), NeuN (red) and DAPI (blue). d Left: representative images of primary neuronal cultures treated with CT-ACM and 22L-ACM for 72 h and co-immunostained for MAP2 (green) and NeuN (red). Right: quantification of neuronal morphology using MAP2 fluorescence in primary neuronal cultures treated with CT-ACM and 22L-ACM for 72 h. e Analysis of expression of Syp, Syn2, Dlg4, Thbs2, Gria1 and Gria4 in primary neuronal cultures treated with 22L-ACM for 72 h and normalized by the expression levels in cultures treated with CT-ACM using qRT-PCR. f Cell viability in primary neuronal cultures assessed by MTT assay as a function of incubation time with CT-ACM or 22L-ACM. In panels a and e, Gapdh was used as housekeeping gene. In panels. a, d–f, data represent means ± SE, n = 3 independent astrocyte or microglia cultures isolated from individual animals, ***p < 0.001, **p < 0.01, *p < 0.05, and ‘ns’ non-significant (two tailed, unpaired student t test). Scale bar = 50 µm

Fig 3: 22L-derived reactive astrocytes show impairment in supporting neuronal growth. Primary cortical neurons were plated on CT-PACs or 22L-PACs and co-cultured for 10–12 days. a Representative fluorescent images of neuron-astrocyte co-cultures co-immunostained for MAP2 (green) and GFAP (red), and quantification of neurite length. b Representative fluorescent images of cortical neuronal cells co-cultured with CT- PACs or 22L-PACs, and co-immunostained for pre- and post-synaptic markers synaptophysin (SYP, green) and PSD95 (red), respectively, and MAP2 (blue). Co-localization between SYP and PSD95 was analyzed for quantification of synapses in neuronal cells co-cultured with CT-PACs or 22L-PACs (yellow puncta). c Representative images of cortical neuronal cells co-cultured with CT-PACs or 22L-PACs, and co-immunostained with a spine marker Drebrin (red) and MAP2 (green). Quantification of spine size and density in neurons co-cultured with CT-PACs and 22L-PACs. In a–c, data represent mean ± SE, n = 3 independent experiments in which CT-PACs or 22L-PACs were isolated individual animals, 50 neurons (in a and b) and 40 neurons (in c) per conditions were analyzed, **p < 0.01 and *p < 0.05 (two tailed, unpaired t test with Welch's correction). Scale bars = 50 µm in a, 25 µm in b, and 10 µm in c