D1R-Mediated Activation of ERK in the Hippocampus Figure 7. Effect of CB1 receptor activation on ERK, AcH3 and rpS6 phosphorylation. 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 (SKF; 5.0 mg/kg) alone or in combination with the CB1 receptor agonist, CP 55,940 (0.25 mg/kg, i.p.). Scale bar: 40 mm. (B, D and F) Quantification of P-ERK, P-AcH3 and P-rpS6 immunoreactivity in mice treated with vehicle, SKF alone or in combination with CP 55,940. Data (means 6 SEM; n = 2–4 mice per group) were analyzed using one-way ANOVA: P-ERK (F(2,5) = 149.01, p,0.001); P-AcH3 (F(2,6) = 13.79, p,0.01); P-rpS6 (F(2,9) = 30.08, p,0.001). Bonferroni-Dunn test: ** p,0.01, *** p,0.001 vehicle vs. SKF, uu p,0.01, uuu p,0.001 vs. SKF. doi:10.1371/journal.pone.0019415.g007 ERK phosphorylation that last for hours [16,17,21]. Overall, the transient activation of ERK induced by SKF 81297 corresponds to the number and duration of seizure events, which peak at 30 min following SKF 81297 administration and decline by 60 min. A more precise analysis of the time at which the first seizures are generated suggests that maximal ERK phosphorylation, which occurs within 15 min after drug administration, may precede the onset of epileptiform activity, which occurs at 20–25 min after drug administration. However that ERK plays a role in the generation of seizures is challenged by the observation that administration of SL327, which abolishes ERK phosphorylation, does not affect pilocarpine-induced seizures [21]. Therefore, increased ERK signaling in the DG most likely represents a marker of neuronal activation indicative of D1R agonist-induced seizures, rather than the cause of such seizures. Interestingly, recent work in a mouse model of pilocarpine-induced temporal lobe epilepsy proposes that phosphorylated ERK may represent an early indicator of activated neurons during spontaneous seizures [23]. Furthermore, the spatio-temporal patterns of ERK phos- phorylation associated with D1R-mediated seizures and sponta- neous (i.e. epileptic) seizures are very similar, suggesting that these two phenomena may recruit the same hippocampal circuits. PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e19415