D1R-Mediated Activation of ERK in the Hippocampus Figure 6. SKF 81297 induces histone H3 phospho-acetylation and rpS6 phosphorylation in the dentate gyrus. (A) Single confocal sections showing immunofluorescence detection of phospho-Ser10-acetyl-Lys14 histone H3 (P-AcH3, upper panel) and phospho-rpS6 (P-rpS6, lower panel) in mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.). Scale bar: 40 mm. (B and C) Quantification of P-AcH3 and P-rpS6 immunoreactive neurons in time course study performed in mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.). Data (means 6 SEM; n = 3 mice per group) were analyzed using one-way ANOVA: P-AcH3 (F(5,12) = 148.78; p,0.001); P-rpS6 (F(5,20) = 33.45; p,0.001). (D and E) Quantification of P-AcH3 and P-rpS6 immunoreactive neurons after pharmacological antagonism with the D1-like family antagonist SCH 23390 (0.15 mg/kg, i.p.). Data (means 6 SEM; n = 4 mice per group) were analyzed using two-way ANOVA: P-AcH3, effect of SKF 81297 (F(1,9) = 67.38; p,0.001), SCH 23390 (F(1,9) = 55.25; p,0.001), interaction (F(1,9) = 57.04; p,0.001); P-rpS6, effect of SKF 81297 (F(1,7) = 352.36; p,0.001), SCH 23390 (F(1,7) = 383.77; p,0.001), interaction (F(1,7) = 402.32; p,0.001). (F and G) Quantification of P-AcH3 and P-rpS6 immunoreactive neurons in wildtype and D1R knock out (D1R KO) mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.). Data (means 6 SEM; n = 2–4 mice per group) were analyzed using two-way ANOVA: P-AcH3, effect of genotype (F(1,8) = 56.01; p,0.001), treatment (F(1,8) = 59.37; p,0.001), interaction (F(1,8) = 58.24; p,0.001); P-rpS6, effect of genotype (F(1,6) = 360.91; p,0.001), treatment (F(1,6) = 313.63; p,0.001), interaction (F(1,6) = 321.28; p,0.001). Bonferroni-Dunn test: *** p,0.001 vehicle vs. SKF 81297, uuu p,0.001 vehicle vs. SCH 23390 or wildtype vs. D1R KO. doi:10.1371/journal.pone.0019415.g006 at the dual site Ser235/236 [20]. Mice given 5.0 mg/kg SKF 81297 showed a rapid, transient increase in the number of phospho-S6 immunoreactive neurons selectively in the granule cells of the DG (Fig. 6A, lower panel and C). This effect of SKF 81297 was abolished by 0.15 mg/kg SCH 23390 (Fig. 6E) and by deletion of D1Rs (Fig. 6G). Activation of CB1 receptors abolishes SKF 81297-induced seizures and reduces phosphorylation of ERK, AcH3 and rpS6 Activation of CB1 receptors, which are abundantly expressed in the hippocampus, has been shown to reduce KA-induced seizures [5,6]. CP 55,940 (0.25 mg/kg) antagonized SKF 81297 (5.0 mg/ kg)-induced phosphorylation of ERK (Fig. 7A and B), AcH3 (Fig. 7C and D) and rpS6 (Fig. 7E and F) in the granular cells of the DG. Thus endocannabinoids, through activation of CB1 receptors, appear to exert an anticonvulsant effect against D1R- induced seizures. SKF 81297-induced phosphorylation of AcH3 and rpS6 depends on ERK activation Parallel increases in the state of phosphorylation of ERK, histone H3 and rpS6 produced by administration of SKF 81297 suggest a causal link between these effects. To examine this possibility we used SL237, an inhibitor of mitogen-activated protein kinase/ERK kinase (MEK) which blocks the phosphory- lation/activation of ERK. The increase in ERK phosphorylation induced by 5.0 mg/kg SKF 81297 was antagonized by a 60 min pretreatment with 50 mg/kg SL327 (Fig. 8A and B). The blockade of ERK phosphorylation was accompanied by antagonism of phosphorylation of AcH3 (Fig. 8C and D) and P-rpS6 (Fig. 8E and F). SKF 81297 promotes the expression of Arc/Arg3.1 and Zif268 via activation of ERK We next investigated whether systemic activation of D1Rs is accompanied by induction of immediate early genes (IEGs) in the hippocampus by determining the effect of 5.0 mg/kg SKF 81297 on levels of Arc/Arg3.1, Zif268 and c-Fos. SKF 81297 induced a large increase in expression of Zif268 and Arc/Arg3.1, specifically in the granule cell layer of the DG (Fig. 9A); Zif268 expression increased at 30 min, peaked at 60 min and returned to basal levels 360 min after SKF 81297 administration (Fig. 9B). The increase in Arc/Arg3.1 expression was more transient; Arc/Arg3.1 expression was first observed at 60 min and returned to basal level 180 min after SKF 81297 administration (Fig. 9C). Increases in Arc/Arg3.1 and Zif268 expression induced by SKF 81297 (5.0 mg/kg) were abolished by pre-treatment with 50 mg/kg SL327 (Fig. 9D, E and F). In contrast, SKF 81297 administration induced only a very modest increase in c-Fos expression in dentate gyrus which did not reach statistical significance when using the Bonferroni post-hoc test (Figure S2). Discussion This study describes a series of unique molecular changes occurring in the hippocampal formation in association with the emergence of epileptiform activity induced by activation of D1Rs. These changes are distinct from those produced by acute injection of chemoconvulsants, such as pilocarpine or KA, and involve increased activation of ERK specifically in the cytoplasm and nuclei of granule cells, leading to phosphorylation of histone H3 and rpS6, and enhanced expression of Zif268 and Arc/Arg3.1. It has been shown that D1R agonists synergise with subthresh- old doses of pilocarpine to induce SE [7]. In addition, it has been reported that, in the mouse, administration of the D1-type receptor agonist SKF 83822 generates seizures [8,9]. This pro- convulsant effect requires intact D1Rs, but not D5Rs, and involves activation of cAMP signaling. In addition, western blot analysis revealed that SKF 83822-induced epileptiform activity was paralleled by a large increase in phospho-ERK immunoreactivity in the hippocampus [9]. Increased ERK phosphorylation in the hippocampal formation is a common response induced by chemoconvulsants. Adminis- tration of pilocarpine at doses able to induce SE and spontaneous seizures increases ERK phosphorylation in the DG, including the hilar region, and, to a lesser extent, in the CA1 [16,21]. In this study, we show that administration of the D1-type receptor agonist SKF 81297 is also able to enhance phospho-ERK immunoreac- tivity in the DG. However, we do not observe any increase in ERK phosphorylation in the CA3 and CA1 pyramidal neurons. Moreover, SKF 81297 does not increase ERK phosphorylation in the interneurons of the hilar region that are affected by administration of pilocarpine and KA [16,22]. Thus, it appears that the degree of activation produced by SKF 81297 in the DG is lower than that necessary to model temporal lobe epilepsy. This is in line with the inability of SKF 81297 to generate SE, neuronal damage or later spontaneous seizures. The increase in ERK phosphorylation produced by SKF 81297 is abolished by genetic inactivation of D1Rs, which also prevents the ability of another D1-type receptor agonist, SKF 83822, to induce epileptiform activity [8,9]. The existence of a link between D1R-mediated induction of seizures and ERK signaling is further suggested by the observation that administration of the D1-type receptor antagonist SCH 23390, or the cannabinoid CB1 agonist CP 55,940, which prevent the manifestation of epileptiform activity, also prevent the ability of SKF 81297 to activate ERK. The increase in ERK phosphorylation produced in the DG by administration of SKF 81297 is short-lived and disappears within 60 min after drug administration. This contrasts with the effects of pilocarpine and KA, whose administration leads to increases in PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19415