D1R-Mediated Activation of ERK in the Hippocampus Figure 4. SKF 81297-induced ERK phosphorylation occurs at the postsynaptic level. (A and B) Single confocal sections showing P-ERK) (red) and MAP2 (green, upper panel) or VAMP2 (green, lower panel) immunofluorescence in the dentate gyrus of mice 15 min after SKF 81297 (5.0 mg/kg, i.p.). Note that P-ERK immunoreactive neurons are observed in MAP2-positive cells, suggesting that ERK activation induced by SKF 81297 occurred mainly at the postsynaptic level. Scale bar: 20 mm. doi:10.1371/journal.pone.0019415.g004 response [18]. Mice given 5.0 mg/kg SKF 81297 showed a robust increase in the number of phospho-acetyl-H3 (P-AcH3) immuno- reactive neurons, restricted to the granule cell layer of the DG (Fig. 6A, upper panels and B); P-AcH3 was maximal 30 min after SKF 81297 administration and returned to basal levels within 3 hr (Fig. 6A and B). This effect of SKF 81297 was abolished by 0.15 mg/kg SCH 23390 (Fig. 6D) and by deletion of D1Rs (Fig. 6F). We next examined the state of phosphorylation of rpS6, a component of the 40S ribosomal subunit [19] which can be phosphorylated by ERK via the 90 kDa ribosomal S6 kinases (RSK) Figure 5. D1Rs are required for SKF 81297-induced ERK phosphorylation in the dentate gyrus. (A) Immunohistochemical detection of P- ERK in the granule cell layer of mice treated with vehicle, SKF 81297 (SKF; 5.0 mg/kg, i.p.) alone or in combination with SCH 23390 (0.15 mg/kg, i.p.). Scale bar: 40 mm. (B) Quantification of P-ERK immunoreactive neurons in the dentate gyrus of mice treated with vehicle and SKF 81297 alone or in combination with SCH 23390 (0.15 mg/kg, i.p.). Data (means 6 SEM; n = 2–4 mice per group) were analyzed using two-way ANOVA: effect of SKF 81297 (F(2,11) = 77.09; p,0.001), SCH 23390 (F(1,11) = 347.53; p,0.001), interaction (F(2,11) = 77.97; p,0.01). (C) Immunohistochemical detection of P- ERK in the granule cell layer of wildtype or D1R knock out (D1R KO) mice treated with vehicle or SKF 81297. Scale bar: 40 mm. (D) Quantification of P- ERK immunoreactive neurons in the dentate gyrus of wildtype or D1R knock out (D1R KO) mice treated with vehicle or SKF 81297. Data (means 6 SEM; n = 2–3 mice per group) were analyzed using two-way ANOVA: effect of genotype (F(1,6) = 48.21; p,0.001), treatment (F(1,6) = 44.11; p,0.001, interaction (F(1,6) = 40.19; p,0.001). Bonferroni-Dunn test: ** p,0.01, *** p,0.001 vehicle vs. SKF 81297; uuu p,0.001 vehicle vs. SCH 23390 and wildtype vs. D1R KO. doi:10.1371/journal.pone.0019415.g005 PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19415