Figure 1.
Progressive DA neurodegeneration depended on the presence of microglia. A, Time course dependence for the effect of LPS, MPP+, and rotenone on DA uptake capacity in the mesencephalic neuron–glia cultures but not in the neuron-enriched cultures. Cultures were treated with vehicle, 10 ng/ml LPS, 0.5 μm MPP+, or 10 nm rotenone for 2–8 d and then assayed for [3H]DA uptake. After 2, 4, 6, and 8 d posttreatment, the amount of DA uptake in vehicle-treated neuron–glia cultures was 0.79 ± 0.14, 0.81 ± 0.04, 0.690 ± 0.10, and 0.60 ± 0.06 pmol/min per well, respectively; in vehicle-treated neuron-enriched cultures, it was 0.88 ± 0.06, 0.86 ± 0.15, 0.82 ± 0.10, and 0.79 ± 0.11 pmol/min per well, respectively. B, Treatment of neuron-enriched cultures with a high dose of rotenone (25 nm) caused a quick and dramatic, but nonprogressive, reduction in DA uptake. C, D, Removal of activated microglia rescued DA neurons from progressive degeneration. The neuron-enriched cultures (N) and reconstituted cultures (N + M) containing microglia (5 × 104 microglia/well) in transwells and enriched neuron layer underneath were treated with vehicle or 10 ng/ml LPS for 24 h. The transwells with treated microglia were then removed from the original culture plates (N + M − M) and put into sister culture plates with existing neuron-enriched cultures (N + treated M). The survival of DA neurons was assessed by [3H]DA uptake immediately (D) or 6 d later (C). The results are expressed as a percentage of the time-matched control cultures and are the mean ± SEM of three to four experiments performed in triplicate. *p < 0.05 compared with the time-matched vehicle-treated controls (A, B) or the corresponding vehicle-treated controls (C, D). p < 0.05 was considered statistically significant. Rot, Rotenone; M, microglia; N + M − M + W, wash the neuron layer after the removal of transwells; N.S., not significant.