Fig. 3
Stat3 deletion alleviates the synaptotoxic effects of reactive astrocytes from prion-infected mice. Cre+/−, 22L-Cre+/−, and TAM-pretreated 22L-Cre+/− astrocytes were co-cultured with primary cortical neurons for 10–12 days. 24 h prior of coculturing with neurons, the culture media containing TAM in astrocyte cultures was replaced with the fresh co-culture media without TAM, as described in Methtods. (A) Left panels: fluorescence microscopy images of cortical neurons co-cultured with Cre+/−, 22L-Cre+/− or TAM-pretreated 22L-Cre+/− astrocytes and co-immunostained for MAP2 and pre- and post-synaptic markers synaptophysin (SYP) and PSD95, respectively. Arrows indicate puncta of co-localized SYP and PSD95. Right panel: quantification of co-localized puncta per field of view in co-cultures. N = 23 random fields of view with 1–2 neurons per field of vies from N = 3 independent cultures. (B) Analysis of expression of Syp, Syn2, Dlg4 and Thbs2 genes in neurons co-cultured with astrocytes using qRT-PCR. C, D. Representative Western blots and densitometric analysis of SYP (C) and PSD-95 (D) expression normalized per expression of β-actin in co-cultures. E. Left panels: fluorescence microscopy images of cortical neuronal cells co-cultured with Cre+/−, 22L-Cre+/− and TAM-pretreated 22L-Cre+/− astrocytes co-immunostained for spine marker Drebrin and MAP2. Right panel: quantification of spine density in co-cultures. In E, 30–40 neurons for each experimental condition. In A and E SuperPlots: colors represent independent experiments; dots represent in individual fields of view or neurons; average values for each experiment are shown as large circles; statistical analyses were performed based on the number of independent experiments; black lines mark means. F. Representative Western blots and densitometric analysis of Stat3 expression astrocytes from adult, non-infected Cre+/− mice, TAM-treated 22L-Cre+/− astrocytes and mock-treated 22L-Cre+/− astrocytes normalized per expression of β-actin. In A-F, Data represent means ± SE, *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA with Bonferroni post-hoc test, N = 3 independent culture experiments per group, each prepared from an individual animal, per group. Scale bars = 25 μm (A) and 10 μm (E)