tide hydrolysis, fails to induce seizures [8]. Furthermore, SKF 83822-induced seizures are reduced by deletion of the dopamine- and cAMP-regulated phosphoprotein-32 kDa (DARPP-32) [9], a critical component of the cAMP signaling machinery implicated in D1R-mediated transmission [10]. However, the neural substrate(s) implicated in dopamine-induced seizures and the biochemical events associated with this phenomenon are still poorly understood. In this study, we examined the ability of SKF 81297, a conventional D1-type receptor agonist, to generate epileptiform activity and affect signal transduction in the hippocampus. Our results indicate that systemic administration of SKF 81297 evokes acute seizures, which do not develop into SE or cause neuronal cell death, and that this effect is associated with a rapid and transient activation of the extracellular signal-regulated protein kinases 1 and 2 (ERK) pathway, specifically in the dentate gyrus (DG). By controlling nuclear and cytoplasmic downstream targets as well as a specific pattern of immediate early gene expression, the activation of the ERK pathway may promote some forms of activity-dependent plasticity in the dentate gyrus related to activation of D1Rs. Materials and Methods Mice Male C57BL/6 mice were obtained at 8 weeks from Taconic (Tornbjerg, Denmark) and Harlan (Bicester, UK). Mutant mice with deletion of the Drd1a gene on an F2 hybrid (1296C57BL/6J) background were generated as described previously (Drago et al., 1994) and backcrossed to wildtype C57BL/6J for 5 generations. Analysis of isolated genomic DNA by PCR was used to genotype the progeny of heterozygous D1R mutant intermatings. Animals were housed in groups of five under standardized conditions with a 12 h light/dark cycle, stable temperature (20uC), and controlled humidity (40–50%). All studies were performed in accordance with the European Communities Council Directive 86/609/EEC for the care and use of experimental animals. These studies were approved by the Research Ethics Committee of Karolinska Institutet and the Royal College of Surgeons in Ireland. They were conducted under licence from the Swedish Animal Welfare Agency and the Department of Health & Children, Ireland, in accordance with the European Communities Council Directive 86/609/EEC for the care and use of experimental animals. Drugs All drugs other than SL327 were purchased from Tocris (Bristol, UK) and injected i.p. in a volume of 10 ml/kg, for biochemical studies, or 4 ml/kg, for behavioural and electrophys- iological studies. For biochemical studies, SKF 81297 and SCH 23390 were dissolved in 0.9% (w/v) saline. For electrophysiolog- ical studies SKF 81297 was dissolved in 0.5% DMSO and SCH 23390 in PCR-grade water. CP 55,940 and SL327 (a-[amino[(4- aminophenyl)-thio]-methylene]-2-(trifluoromethyl)-benzeneaceto- nitrile; an inhibitor of the mitogen-activated protein kinase/ERK kinase (MEK), which blocks the phosphorylation/activation of ERK), a gift from Dr. Edilio Borroni (Hoffmann-La Roche, Basel, Switzerland), were suspended by sonication in a solution of 8% Tween 80 in saline and administered i.p. 15 and 60 min before SKF 81297 injection, respectively. Control animals received equivalent volumes of the respective vehicles. Behavioural assessment For evaluation of drug-induced seizures, mice were removed from their home cage and placed individually in clear glass observation cages (38638625 cm) with wood shavings as bedding D1R-Mediated Activation of ERK in the Hippocampus material. Mice were then allowed to habituate for a period of 3 h to reduce initially high levels of activity in order to optimize detection of agonist-induced behavioural effects. Immediately following challenge with SKF 81297 (0.5–5.0 mg/kg) or vehicle, mice were recorded using digital video for 60 min. Video recordings was subsequently scored for seizures, characterized as any of the following phases occurring within the 60 min recording period: phase 1 = transient ($5 sec) disruption of exploratory behaviour by tonic immobility/rigidity; phase 2 = rearing with forepaw myoclonus; phase 3 = generalized clonus; phase 4 = tonic- clonic seizure or rapid jumping and wild running (O’Sullivan et al. 2008); all recordings were scored by an observer who was unaware of the treatment of each animal. The D1R-type antagonist SCH 23390 (0.15 mg/kg) or the cannabinoid CB1 agonist CP 55,940 (0.25 mg/kg) were administered 5 and 15 min, respectively, prior to administration of SKF 81297. Mice were used once only. Electroencephalographic recording Seizure studies were carried out in a manner similar to that described previously [9]. Briefly, for surgical implantation of extradural and intrahippocampal recording electrodes, mice were initially anaesthetised using 5% isoflurane (Abbott Labo- ratories, Kent, UK) in O2, placed in a stereotaxic frame and maintained under anaesthesia using 1.5% isoflurane in O2. Temperature was maintained normothermic (3760.5uC) by means of a rectal thermometer and thermostatically controlled heating pad (Harvard Apparatus, MA, USA). The scalp was incised and a burr hole drilled in the exposed cranium over the hippocampus and the frontal cortex. A recording electrode and a frontal reference electrode (Plastics One Inc., VA, USA) were then implanted. A third complete craniotomy was drilled for the placement of a twisted bipolar electrode (Plastics One Inc., VA, USA) into the right dentate gyrus (coordinates from Bregma: Figure 1. SKF 81297 induces behavioural seizures. Counts for seizure behaviours (any of phase 1–4 seizure phenotypes; see Materials and Methods) in mice treated with vehicle, SKF 81297 (0.5–5.0 mg/kg, i.p.) or SKF 81297 (5.0 mg/kg, i.p.) following administration of SCH 23390 (0.15 mg/kg, i.p.) or CP 55,940 (0.25 mg/kg, i.p.). Data (means 6 SEM; n = 8 mice per group) were analysed using Kruskal-Wallis nonparametric ANOVA: dose-response for induction of seizures by SKF 81297 (p,0.05); Mann-Whitney U-tests: * p,0.05 vehicle vs. SKF 81297; u p,0.05 SKF 81297 vs. SKF 81297 following pre-treatment with SCH 23390 and SKF 81297 following pre-treatment with CP 55,940. doi:10.1371/journal.pone.0019415.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19415