Figure 1

Characterization and effects of morphine and/or HIV-1 Tat exposure on bound and soluble fractalkine, and CX ₃ CR1 levels in mixed striatal neuron-glia cultures. Fractalkine immunofluorescence was readily co-localized in MAP2-positive neuron cell bodies and dendrites (A), while a majority of the CX₃CR1 immunoreactive cells were CD11b-positive microglia (B); scale bar = 15 μm. Fractalkine derived from neuron-glial co-cultures presented a prominent band at approximately 95-kDa (C). This is slightly higher than the non-glycosylated, recombinant fractalkine (rFKN) control protein detected at 90-kDa; 0.1 μg and 0.025 μg fractalkine (FKN) were loaded onto the left-hand lanes. The relative abundance of either variant appeared to be unaffected by morphine (Morph) and/or Tat treatment at 24 h (C); n = 3 experiments. The amount of fractalkine released into the culture medium was unaffected following 2 h of morphine and/or Tat exposure (D); the limit of fractalkine detection by ELISA was 320 pg/ml (standard curve in D); n = 3 experiments. Unlike fractalkine, treatment with morphine and Tat together significantly reduced CX₃CR1 levels (~47-kDa) at 24 h, but not 6 h or 12 h, compared to vehicle-treated controls (E) (*P < 0.02 vs. vehicle-treated controls; Kruskal-Wallis ANOVA and nonparametric post hoc tests), while CX₃CR1 levels were unaffected by exposure to morphine or Tat alone (E); fractalkine and CX₃CR1 levels were normalized to actin (~43-kDa bands in C,E) in immunoblots; n = 6 experiments.