Fig. 7

TPF^PD-stimulated microglial cells trigger more excitotoxic cell death than F^PD-exposed cells on dopaminergic neurons. A Schematic representation of the experimental setup used to analyze the neurotoxic potential of chronic-type inflammatory microglia. Microglial cell cultures were maintained in DMEM^S-based astrocyte-conditioned medium (ACM) and exposed or not (NSC) for 6 h to either F^PD or TPF^PD. Following inflammatory stimulation, the culture medium was fully replaced with fresh DMEM^S-based ACM (washout step), and the microglial cells were left for 24 h before the glutamate assay (B). Midbrain neuronal cultures were prepared from E13.5 mouse embryos and maintained for 2 days in Nb^S. On day in vitro 2 (DIV2), the culture medium was fully replaced with Nb^S-based ACM and the neurons were left to mature until DIV6. At DIV6, 30% (v/v) of the Nb^S-based ACM media was replaced with microglia-conditioned media (MCM) and dopaminergic neuron survival was assayed 24 h later. The data are presented as the means ± SEM (biological replicates n = 13; 3 independent experiments). ***p < 0.001 vs NSC and F^PD (One-way ANOVA followed by Tukey’s test). C Low- and high-magnification (dashed brown squares) images of tyrosine hydroxylase (TH) immunostained DIV7 midbrain cultures exposed for 24 h to MCM from F^PD and TPF^PD-stimulated or unstimulated cells (NSC). Brown arrows point to dystrophic cell bodies and processes with varicosities. Scale Bar = 100 µm. D Quantification of TH+ neurons in DIV7 midbrain cultures exposed for 24 h to MCM from F^PD and TPF^PD-stimulated or unstimulated cells (NSC). In sister cultures, neurons were exposed to MCM from F^PD and TPF^PD-stimulated microglial cells treated with the xCT inhibitor Sulfasalazine (250 µM) during the 24 h washout period. In additional sister cultures, neurons exposed to MCM from F^PD and TPF^PD-stimulated microglial cells were cotreated with the N-methyl-D-aspartate (NMDA) receptor pore blocker MK-801 (2 µM). The data are expressed as a % of neurons grown in Nb^S only and are presented as the means ± SEM (biological replicates n = 5–8; 2 independent experiments). *** p < 0.001 vs. NSC or F^PD (One-way ANOVA followed by pairwise multiple comparisons using the Holm-Sidak method). E TH+ dopaminergic neuron survival is inversely correlated with the concentration of glutamate within the transferred MCM (linear regression analysis)