D1R-Mediated Activation of ERK in the Hippocampus Figure 3. SKF 81297 induces ERK phosphorylation in the dentate gyrus. (A) Single confocal section showing phosphorylated ERK (P-ERK) in the hippocampus of mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.). Scale bar: 150 mm. (B) Dose-response (0, 0.5, 1, 2.5, and 5.0 mg/kg) and (C) time course (0, 15, 30, 60 and 180 min) of P-ERK in mice treated with SKF 81297. Scale bar: 40 mm. (D and E) Quantification of phosphorylated ERK (P-ERK) immunoreactive neurons in the dentate gyrus of mice treated with vehicle or SKF 81297. Data (means 6 SEM; n = 3–4 mice per group) were analyzed using one-way ANOVA: dose-response (F(4,9) = 61.22; p,0.001), time course (F(5,12) = 239.78; p,0.001). (F) Single confocal section showing P- ERK in the CA3 and CA1 areas of mice treated with vehicle or SKF 81297 (5.0 mg/kg, i.p.). Scale bar: 40 mm. (G) Quantification of P-ERK immunoreactive neurons in CA3 and CA1 of mice treated with vehicle or SKF 81297. Data (means 6 SEM; n = 4–5 mice per group) were analyzed using student t-test: CA3 (F(1,7) = 1.23; NS), CA1 (F(1,7) = 0.65; NS). doi:10.1371/journal.pone.0019415.g003 Beverly, MA, USA) and mouse monoclonal antibody for MAP2 (1:1000, Sigma-Aldrich, Sweden) overnight at 4uC. Antibodies for c-Fos (1:400), Arc/Arg3.1 (1:400), and Zif268 (1:400) were purchased from Santa Cruz biotechnology and were incubated with tissue sections for 72 h at 4uC in a solution of BSA 1%. After three rinses in TBS, sections were incubated for 45 min at room temperature (RT) with goat-anti rabbit Cy3-coupled (1:500) and goat-anti mouse Cy2-coupled (1:500) secondary antibodies (Jackson ImmunoResearch Europe Ltd). A monoclonal antibody against VAMP2 directly coupled to Alexa 488 was used (1:2000; gift from Dr. Peter Lo¨w, Karolinska Institutet) [13]. Sections were then rinsed twice in TBS and twice in TB and mounted on a slide under cover slips using 1,4-diazabicyclo-[2. 2. 2]-octane (DABCO, Sigma-Aldrich Sweden AB, Stockholm). Fluoro-JadeH (Millipore Corp., Billica, MA, USA) staining was used to label degenerating neurons, as previously described [14]. Analysis of immunofluorescence Bilateral images were obtained using sequential laser scanning confocal microscopy (Zeiss LSM 510, Jena, Germany) and quantified using ImageJ software (National Institutes of Health). Quantification was performed in 325.75 mm6325.75 mm images by counting Cy2- and Cy3-immunofluorescent cells. Statistical analysis Behavioural seizure events were summed over the 60 min recording period, expressed as means 6 SEM and analysed using the Kruskal-Wallis non-parametric ANOVA, followed by Mann- Whitney U-test. Immunofluorescence data were expressed as means 6 SEM and analyzed by one-way or two-way ANOVA followed by Bonferonni-Dunn test. Statistical analysis was performed using StatView software, with p,0.05 adopted as the threshold for significance. Results Seizure profile in response to systemic administration of SKF 81297 SKF 81297 (0.5–5.0 mg/kg) dose-dependently induced behav- ioural seizures (p,0.05; Fig. 1), which were observed in 6 (75%) of 8 mice receiving 5.0 mg/kg. Among the 4 phases of seizure activity recorded (see materials and methods), there was no significant preponderance of any seizure phenotype. Behavioural seizures induced by 5.0 mg/kg SKF 81297 were abolished by pretreatment with either 0.15 mg/kg SCH 23390 or 0.25 mg/kg CP 55,940 (each p,0.05; Fig. 1). Simultaneous EEG recordings from skull-mounted surface electrodes and intra-cerebral depth recordings from the DG following injection of SKF 81297 (5.0 mg/kg) demonstrated the presence of seizures (high amplitude and high frequency discharges) in the DG in 5 (71%) of 7 mice; mean latency to emergence of seizures was 2262 min. There was a time- dependent increase in seizure incidence and duration following injection of SKF 81297 (Fig. 2), with seizure severity peaking at 30–45 min post-injection and declining by 60 min (Fig. 2B, C). No seizure activity was evident in vehicle-treated mice (n = 5; data not shown). The SKF 81297-induced seizures were not accompanied by the appearance of neurodegenerative responses, as determined by Fluoro-Jade staining, 6 hrs after recording (data not shown). SKF 81297-induced seizures are associated with an increase of ERK phosphorylation selectively in the dentate gyrus of the hippocampus Pharmacological induction of seizures by KA and pilocarpine is accompanied by increased ERK phosphorylation in several hippocampal regions [15,16,17]. Therefore we examined the induction and distribution of phospho-ERK (P-ERK) in the hippocampus of mice treated with SKF 81297. Administration of 2.5 and 5.0 mg/kg SKF 81297 resulted in a large increase in P- ERK immunoreactivity selectively in the granule cell layer of the DG (Fig. 3A, B and D). This effect peaked at 15–30 min post- injection and declined by 60 min (Fig. 3C and E). P-ERK immunoreactivity was detected in neuronal nuclei and perikarya, as well as in the dendrites of almost all the granule cells of the DG (Fig. 3A–C). In contrast, administration of 5.0 mg/kg SKF 81297 did not affect ERK phosphorylation in the CA3 and CA1 pyramidal neurons (Fig. 3F and G). Dendritic activation of ERK was confirmed by the strong co-localization of P-ERK with the microtubule-associated protein 2 (MAP2), a protein highly enriched in dendrites (Fig. 4A). In contrast, no co-localization of P-ERK immunoreactivity was found with a marker of nerve terminals, the vesicle-associated membrane protein 2 (synapto- brevin 2, also known as VAMP2) (Fig. 4B). The effect of SKF 81297 (5 mg/kg) on ERK phosphorylation was compared to those produced by the D1-type agonists SKF 83822 and SKF 83959, which are considered to preferentially activate cAMP and phospholipase C signaling, respectively [9]. We found that SKF 83822 (2 mg/kg) produced an increase in the number of P-ERK positive neurons comparable to that of SKF 81297 (Figure S1). The same dose of SKF 83959 was also able to increase ERK phosphorylation, however its effect was significantly lower than those produced by SKF 81297 or SKF 83822 and appeared to be preferentially exerted in the cytoplasm (Figure S1). SKF 81297-induced ERK phosphorylation is prevented by inactivation of dopamine D1 receptors The increase in ERK phosphorylation produced by 2.5 or 5.0 mg/ kg SKF 81297 was abolished by 0.15 mg/kg SCH 23390 (Fig. 5A and B) and was absent in mice with deletion of D1Rs (Fig. 5C and D). These results indicate that SKF 81297-induced seizures increase ERK phosphorylation in the DG through stimulation of D1Rs. SKF 81297 induces phospho-acetylation of histone H3 and phosphorylation of rpS6 We proceeded by determining whether SKF 81297-induced seizures enhanced phosphorylation of histone H3, an important downstream nuclear target of ERK involved in the nucleosomal PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19415