FIGURE 3.

Iron repletion or choline supplementation restores dendrite complexity of iron-deficient neurons. Hippocampal neurons cultured from embryonic day 16 (E16) mice were nucleofected with an eGFP-tagged PSD95 intrabody at plating. Iron deficiency was induced beginning at 3 d in vitro (DIV) by treating with 9 μM deferoxamine (DFO). At 11 DIV, DFO was removed to begin iron repletion and 30 μM choline chloride was added to begin choline supplementation. (A) Representative 18 DIV images of neurons expressing the PSD95 intrabody (top row) after immunocytochemistry for MAP2 (second row). Merged images (third row) were used to trace the dendritic arbors (bottom row) for green fluorescent protein–positive neurons. (B) Sholl analysis (mean ± SEM; n = 61–100 neurons). Note: X–Y data variances are shown as SEMs rather than SDs for easier visualization. For a given distance from the soma, colored asterisks (*) indicate a statistical difference compared with the ID group by repeated-measures 2-factor ANOVA and Tukey test (P < 0.05). The full statistical comparisons are shown in Supplemental Table 1. (C) Average total length of the entire dendritic arbor (mean ± SD, n = 61–100 neurons). Groups that do not share a common letter are statistically different by 2-factor ANOVA and Tukey test (P < 0.05). An asterisk (*), if present, indicates a statistically significant effect of iron or choline status. A delta symbol (∂), if present, indicates a statistically significant interaction effect. Cho, choline; eGFP, enchanced green fluorescent protein; FID, formerly iron deficient; ID, iron deficient; IS, iron sufficient.