Fig. 5.

Microglia activation by LPS induces the release of ATP that recruits astrocytes. (A) RNA expression levels relative to GAPDH were used to test the purity of the cultures of astrocytes (white bars) and microglia (black bars). GFAP, Iba1, and Neuroligin 1 transcripts were used as markers of astrocytes, microglia, and neuron, respectively (84). (B) TLR4 and CD14 mRNA are only detected in microglia by qRT-PCR using pure cultures of microglia (black bars) and microglia-free culture of astrocytes (white bars, n = 3 cultures, mean ± SEM, ANOVA, *P < 0.05, **P < 0.01 compared with Nlgn1). (C) Cultured astrocytes identified by GFAP immunostaining (Upper Left) do not bind Alexa-568–tagged LPS (Lower Left). In contrast, Alexa-568 LPS (Lower Right) colocalized with cultured microglia identified by F4/80 immunostaining (Upper Right). (D) LPS application on pure microglial cultures induces the production of ATP when microglia were plated at high concentration (mean ± SEM, ANOVA, **P < 0.01, n = 6 cultures). (E) LPS application to pure confluent astrocytes (Astro; white bars) or pure microglia at low concentration (μglia; 3 × 10⁵ cells/mL; black bars) does not induce the production of ATP. LPS application to mixed cultures containing microglia at a low concentration added to confluent LME-treated astrocyte cultures (Astro + μglia; gray bars) induced the production of ATP (n = 6, average ± SEM). (F) LPS-mediated ATP release in mixed cultures (black bars) was prevented by RB-2 (n = 12 wells, six cultures) or PPADS (n = 10 wells, five cultures, mean ± SEM, **P < 0.01; white bars).