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. 2019 Jul 29;6(4):ENEURO.0237-19.2019. doi: 10.1523/ENEURO.0237-19.2019

Figure 5.

Figure 5.

Morphine and iron upregulate FHC and FLC in cortical neurons. A, Iron-loading upregulates FHC and FLC in neurobasal cultures. Cultures were iron-loaded with FAC (25 µM) for 30 min, 6 h, or 24 h before lysis. Additionally, a negative control culture was iron-chelated with DFO (100 µM, 24 h) before lysis. Iron loading with FAC significantly increased FHC and FLC but only after 24 h. FHC and FLC expression were not significantly different at any time after treatment; N = 3 experiments; treatment F(4,20) = 12.94, p < 0.0001; FHC/FLC expression F(1,20) = 0.0029, p = 0.9576. B, Morphine upregulates FHC and FLC in neurobasal cultures. Cultures were treated with morphine (1 µM) or vehicle and lysed 30 min, 6 h, or 24 h after treatment. Morphine upregulated both FHC and FLC, but FHC was significantly upregulated at 6 h, while FLC reached significance at 24 h. However, the overall expression of FHC was not significantly different from FLC at each time point; N = 4 experiments; treatment F(3,24) = 22.94, p < 0.0001; FHC/FLC expression F(1,24) = 9.252, p = 0.0056. C, Morphine-treated rats upregulate FHC and FLC in frontal cortex tissue. Three-week-old Holtzman rats were treated with extended-release morphine or placebo pellets for 96 h as described in Figure 4 and the Materials and Methods. After the treatment, rats were killed and frontal cortex tissue was dissected, homogenized, and analyzed by Western blotting. Morphine significantly increased FHC and FLC expression in vivo, similarly to the in vitro experiment in panel B; N = 4 rats per treatment group; each column contains a homogenate from a different rat; treatment F(1,12) = 43.94, p < 0.0001; FHC/FLC expression F(1,12) = 3.814, p = 0.0745. All data were analyzed by two-way ANOVA and Tukey post hoc.