Figure 4. CXCL12 regulates dendritic spine density in vitro and in vivo.

(A) Rat cortical neurons were cultured for 21 days in the presence of a glial feeder layer. CXCL12 treatment (20 nM, 3 hours) increased dendritic spine density (n = 12). (B) Rat cortical neurons were cultured for 21 days as a pure neuronal population. CXCL12 treatment (20 nM) time-dependently increased dendritic spines (n = 7–29). (C) CXCL12 treatment (20 nM) also increased nuclear HDAC4 levels in pure neuronal cultures, as visualized by immunocytochemistry at 30 minutes and quantified over time (n = 12–34). Similar results were obtained by Western blot using nuclear extracts from neurons. (D) After 8 days in culture, transfection of a pure neuronal population with siRNA specifically decreased HDAC4, but not HDAC1, 36 hours after transfection. siRNA-mediated HDAC4 deficiency completely blocked the effects of CXCL12 on dendritic spines (n = 8–17). (E) The CXCR4 antagonist AMD3100 (1 μg i.c.v.) was injected into the left lateral ventricle of 3-week-old rats. 6 hours after injection, animals were sacrificed, and layer II/III pyramidal neurons were analyzed, revealing reduced total dendritic spine density (n = 15). (F) This effect of AMD3100 was similarly confirmed in a prolonged treatment model: implantation of 3-week-old rats with osmotic pumps delivering constant levels of AMD3100 (0.75 μg/h) for 4 days decreased total dendritic spine density (n = 15). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 5 μm (A); 10 μm (C, E, and F). See complete unedited blots in the supplemental material.