Figure 3

Studies with Cx3cr1^-/- mice: Fluorescent images and effects of Cx3cr1^-/- glia on fractalkine-mediated neuroprotection. Images of the striatum (A-C) and of mixed glial cultures (D) from Cx3cr1^-/- mouse striata. Microglia within the striatum of Cx3cr1^-/- (Cx3cr1^GFP/GFP) mice fluoresce green (A,B) and are Iba-1 immunoreactive (C); Hoechst counterstained nuclei (blue); scale bars = 50 μm (A) and 10 μm (B,C). Robust GFP expression is maintained when the cells are placed into culture (D). As can be appreciated from the Hoechst stain, the CX₃CR1-expressing population is significant although the majority of the cells in the culture lack CX₃CR1. The loss of CX₃CR1 on glia has a dramatic effect on the ability of fractalkine to protect morphine and Tat-treated striatal neurons (E). Neurons from the striata of wild-type mice were plated onto mixed glia prepared from Cx3cr1^-/- mouse striata and followed over 48 h using our standard paradigm. In general, wild-type neurons co-cultured with Cx3cr1^-/- glia appeared to be less viable than with wild-type glia (upper panel; compare gray and black dotted lines); however, these groups were not compared statistically since the experiments were not run concurrently. The neuroprotective effects of fractalkine (CX₃CL1; 1 μg/ml) are abolished when wild-type neurons are co-cultured with Cx3cr1^-/- mixed glia. Under these conditions, fractalkine no longer protects neurons from combined morphine and Tat exposure (E, lower panel; *P < 0.05 vs. vehicle- or fractalkine-treated controls). There were no differences in survival between controls ± fractalkine. Addition of fractalkine did not change the toxicity of morphine, Tat, or morphine + Tat treatments. There is, however, some possibility that fractalkine enhances baseline neuron survival in the Cx3cr1^-/- glial co-cultures. This was indicated by significant differences only at 48 h between Tat + fractalkine versus control + fractalkine survival (^§P < 0.05), even though survival did not differ when comparing vehicle + control vs. fractalkine + control or vehicle + Tat vs. fractalkine + Tat treatments. A similar inconsistency was observed only at 48 h with morphine treatments (^§P < 0.05).