D1R-Mediated Activation of ERK in the Hippocampus Figure 8. AcH3 and rpS6 phosphorylation induced by SKF 81297 is dependent on ERK activation. Immunohistochemical detection of P- ERK (A), P-AcH3 (C) and P-rpS6 (E) in the granule cell layer of mice treated with vehicle, SKF 81297 (5.0 mg/kg, i.p.) alone or in combination with the MEK inhibitor, SL327 (50 mg/kg, i.p.). Scale bar: 40 mm. (B, D and F) Quantification of P-ERK, P-AcH3 and P-rpS6 immunoreactive neurons in mice treated with SKF 81297 alone or in combination with SL327 (50 mg/kg, i.p.) and perfused 15 and 30 min later. Data (means 6 SEM; n = 4–5 mice per group) were analyzed using two-way ANOVA: P-ERK, effect of time (F(1,10) = 0.61, NS), treatment (F(1,10) = 235.19, p,0.001), interaction (F(1,10) = 2.91, NS); P-AcH3, effect of time (F(1,10) = 8.05, p,0.02), treatment (F(1,10) = 91.73, p,0.001), interaction F(1,10) = 4.27, NS); P-rpS6, effect of time (F(1,17) = 34.54, p,0.001), treatment (F(1,17) = 17.67, p,0.001), interaction (F(1,17) = 0.16, NS). Bonferroni-Dunn test: ** p,0.01, *** p,0.001 vehicle vs. SL327. doi:10.1371/journal.pone.0019415.g008 The activation of ERK produced by administration of SKF 81297 occurs both in the cytoplasm and in the nucleus of granule cells, as shown by the presence of phosphorylated ERK in these two compartments. In line with this observation, we found that activation of D1Rs increased the state of phosphorylation of rpS6 and histone H3, two downstream targets of ERK selectively expressed in the cytoplasm and the nucleus, respectively. Time- course analysis indicated a rapid increase in rpS6 phosphorylation, which peaked at 15 min after administration of SKF 81297, and a more progressive increase in histone H3 phosphorylation, which reached a peak at 30 min and was still significantly elevated at 60 min. Such kinetics of phosphorylation indicates a rapid and more transient activation of ERK signaling in the cytoplasm, paralleled by a slower and more resilient activation in the nucleus. Phosphorylation of rpS6 is likely to occur via activation of RSK, a direct ERK substrate and major rpS6 kinase, phosphorylation of which was also enhanced following SKF 81297 administration (data not shown). However, the increase in rpS6 phosphorylation PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e19415